Compositions and Methods of Use for Treating Metabolic Disorders

ABSTRACT

Methods of treating individuals with a glucose metabolism disorder and/or a body weight disorder, and compositions associated therewith, are provided.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication No. 61/616,294, filed Mar. 27, 2012, which application isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to, among other things, growthdifferentiation factor muteins and modifications thereof which areuseful in treating obesity, diabetes and other metabolic-relateddisorders.

BACKGROUND

Obesity is most commonly caused by excessive food intake coupled withlimited energy expenditure and/or lack of physical exercise. Obesityincreases the likelihood of development of various diseases, such asdiabetes mellitus, hypertension, atherosclerosis, coronary arterydisease, sleep apnea, gout, rheumatism and arthritis. Moreover,mortality risk directly correlates with obesity, such that, for example,a body-mass index in excess of 40 results in an average decreased lifeexpectancy of more than 10 years.

Current pharmacological treatment modalities include appetitesuppressors targeting receptor classes (e.g., CB1, 5-HT_(2C), and NPY);regulators of the appetite circuits in the hypothalamus and themolecular actions of ghrelin; and nutrient-absorption inhibitorstargeting lipases. Unfortunately, none of the current modalities hasbeen shown to effectively treat obesity without causing adverse effects,some of which can be very severe.

High blood glucose levels stimulate the secretion of insulin bypancreatic beta-cells. Insulin in turn stimulates the entry of glucoseinto muscles and adipose cells, leading to the storage of glycogen andtriglycerides and to the synthesis of proteins. Activation of insulinreceptors on various cell types diminishes circulating glucose levels byincreasing glucose uptake and utilization, and by reducing hepaticglucose output. Disruptions within this regulatory network can result indiabetes and associated pathologic syndromes that affect a large andgrowing percentage of the human population.

Patients who have a glucose metabolism disorder can suffer fromhyperglycemia, hyperinsulinemia, and/or glucose intolerance. An exampleof a disorder that is often associated with the aberrant levels ofglucose and/or insulin is insulin resistance, in which liver, fat, andmuscle cells lose their ability to respond to normal blood insulinlevels.

In view of the prevalence and severity of obesity, diabetes andassociated metabolic and non-metabolic disorders, along with theshortcomings of current treatment options, alternative treatmentmodalities that modulate, for example, appetite, glucose and/or insulinlevels and enhance the biological response to fluctuating glucose levelsin a patient remain of interest.

SUMMARY

The present disclosure contemplates the use of the agents describedherein, and compositions thereof, to treat and/or prevent variousdiseases, disorders and conditions, and/or the symptoms thereof. In someembodiments, the diseases, disorders and conditions, and/or the symptomsthereof, relate to glucose metabolism disorders and othermetabolic-related disorders, whereas in other embodiments they relate tobody weight disorders. By way of example, but not limitation, theagents, and compositions thereof, can be used for the treatment and/orprevention of diabetes mellitus (e.g., Type 2 diabetes), insulinresistance and diseases, disorders and conditions characterized byinsulin resistance, decreased insulin production, hyperglycemia,hypoinsulinemia, and metabolic syndrome. The agents, and compositionsthereof, can also be used for the treatment and/or prevention of obesityand other body weight disorders by, for example, effecting appetitesuppression.

In certain embodiments, the agents are human Growth DifferentiationFactor 15 (GDF15)-related polypeptides, and homologs, variants (e.g.,muteins), fragments and other modified forms thereof. In particularembodiments, the agents contemplated by the present disclosure aremodified human GDF15 molecules, whereas in other embodiments the agentsare GDF15 muteins or modified GDF15 muteins. The present disclosure alsocontemplates nucleic acid molecules encoding the foregoing. For the sakeof convenience, the modified human GDF15 molecules, the GDF15 variants(e.g., muteins), and the modified GDF15 variants (e.g., muteins)described henceforward are collectively referred to hereafter as the“Polypeptide(s)”. It should be noted that any reference to “human” inconnection with the polypeptides and nucleic acid molecules of thepresent disclosure is not meant to be limiting with respect to themanner in which the polypeptide or nucleic acid is obtained or thesource, but rather is only with reference to the sequence as it maycorrespond to a sequence of a naturally occurring human polypeptide ornucleic acid molecule. In addition to the human polypeptides and thenucleic acid molecules which encode them, the present disclosurecontemplates GDF15-related polypeptides and corresponding nucleic acidmolecules from other species.

The present disclosure also contemplates other GDF15-related agentscapable of eliciting a biological response comparable to (or greaterthan) that of the Polypeptides, and/or agents capable of enhancing theactivity of the Polypeptides.

In some embodiments of the present disclosure, a subject having, or atrisk of having, a disease or disorder treatable by one or morePolypeptides is administered in an amount effective for treating thedisease or disorder. In some embodiments, the disease or disorder is ahyperglycemic condition, insulin resistance, hyperinsulinemia, glucoseintolerance or metabolic syndrome. In other embodiments the disease ordisorder is a body weight disorder (e.g., obesity), while in still otherembodiments the Polypeptides cause, to at least some extent, appetitesuppression.

Other aspects of the present disclosure include cell-based expressionsystems, vectors, engineered cell lines, and methods and uses related tothe foregoing.

As described in detail hereafter, one embodiment of the presentdisclosure relates to a peptide comprising any one of: a) a peptidecomprising at least one modification to the sequence depicted in FIG. 1B(SEQ ID NO:3); b) a mutein peptide of the sequence depicted in FIG. 1B(SEQ ID NO:3); or c) a mutein peptide of the sequence depicted in FIG.1B (SEQ ID NO:3), wherein the mutein peptide comprises at least onemodification.

In certain embodiments of the present disclosure, a peptide comprises amutein peptide of any one of v1-v23 as depicted in FIG. 2.

In other embodiments, the modification to a peptide comprisespegylation, glycosylation, polysialylation, hesylation, albumin fusion,albumin binding through a conjugated fatty acid chain, Fc-fusion, orfusion with a PEG mimetic. In particular embodiments, the modificationcomprises pegylation.

In still further embodiments, a peptide of the present disclosurecomprises an amino acid sequence having at least 85% amino acididentity, at least 90% amino acid identity, at least 93% amino acididentity, at least 95% amino acid identity, at least 97% amino acididentity, at least 98% amino acid identity, or at least 99% amino acididentity to the amino acid sequence depicted in FIG. 1B (SEQ ID NO:3)

A peptide of the present disclosure may have fewer than 100 amino acidresidues, fewer than 75 amino acid residues, fewer than 50 amino acidresidues, fewer than 25 amino acid residues, or fewer than 20 amino acidresidues.

According to the present disclosure, the peptide may be producedrecombinantly.

Furthermore, the present disclosure contemplates nucleic acid moleculesencoding the aforementioned peptides. In some embodiments, a nucleicacid molecule is operably linked to an expression control element thatconfers expression of the nucleic acid molecule encoding the peptide invitro, in a cell or in vivo. In some embodiments, a vector (e.g., aviral vector) contains one or more of the nucleic acid molecules.

Some embodiments include transformed or host cells that express one ormore of the aforementioned peptides.

In particular embodiments of the present disclosure, one or more of theaforementioned peptides is formulated to yield a pharmaceuticalcomposition, wherein the composition also includes one or morepharmaceutically acceptable diluents, carriers or excipients. In certainembodiments, a pharmaceutical composition also includes at least oneadditional prophylactic or therapeutic agent.

Still further embodiments of the present disclosure comprise an antibodythat binds specifically to one of the aforementioned mutein peptides. Insome embodiments, the antibody comprises a light chain variable regionand a heavy chain variable region present in separate polypeptides or ina single polypeptide. An antibody of the present disclosure binds thepeptide with an affinity of from about 10⁷ M⁻¹ to about 10¹² M⁻¹ incertain embodiments. In still other embodiments, the antibody comprisesa heavy chain constant region of the isotype IgG1, IgG2, IgG3, or IgG4.In additional embodiments, the antibody is detectably labeled, while itis a Fv, scFv, Fab, F(ab′)₂, or Fab′ in other embodiments.

The present disclosure also contemplates antibodies that comprise acovalently linked non-peptide polymer (e.g., a poly(ethylene glycol)polymer). In other embodiments, the antibody comprises a covalentlylinked moiety selected from a lipid moiety, a fatty acid moiety, apolysaccharide moiety, and a carbohydrate moiety.

The antibody is a single chain Fv (scFv) antibody in some embodiments,and the scFv is multimerized in others.

The antibodies of the present disclosure may be, but are not limited to,monoclonal antibodies, polyclonal antibodies, or humanized antibodies.

Furthermore, the present disclosure contemplates pharmaceuticalcompositions comprising an antibody as described above formulated withat least one pharmaceutically acceptable excipient, carrier or diluent.Such pharmaceutical compositions may also contain at least oneadditional prophylactic or therapeutic agent.

Certain embodiments of the present disclosure contemplate a sterilecontainer that contains one of the above-mentioned pharmaceuticalcompositions and optionally one or more additional components. By way ofexample, but not limitation, the sterile container may be a syringe. Instill further embodiments, the sterile container is one component of akit; the kit may also contain, for example, a second sterile containerthat contains at least one prophylactic or therapeutic agent.

The present disclosure also contemplates a method of treating orpreventing a glucose metabolism disorder in a subject (e.g., a human) byadministering to the subject a therapeutically effective amount of aPolypeptide. In some methods, the treating or preventing results in areduction in plasma glucose in the subject, a reduction in plasmainsulin in the subject, or an increase in glucose tolerance in thesubject. In particular embodiments, the glucose metabolism disorder isdiabetes mellitus. In some embodiments, the subject is obese and/or hasa body weight disorder.

Though not limited to any particular route of administration or dosingregimen, in some embodiments the administering is by parenteral (e.g.,subcutaneous) injection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts the human GDF15 precursor amino acid sequence (SEQ IDNO:1) and the corresponding nucleic acid (SEQ ID NO:2) encoding thehuman GDF15 precursor amino acid sequence.

FIG. 1B depicts the mature human GDF15 amino acid sequence (SEQ ID NO:3)and the corresponding nucleic acid sequence (SEQ ID NO:4) encodingmature human GDF15.

FIG. 2 depicts the amino acid sequences of GDF15 muteins v1-v23. v1 (SEQID NO:5); v2 (SEQ ID NO:6); v3 (SEQ ID NO:7); v4 (SEQ ID NO:8); v5 (SEQID NO:9); v6 (SEQ ID NO:10); v7 (SEQ ID NO:11); v8 (SEQ ID NO:12); v9(SEQ ID NO:13); v10 (SEQ ID NO:14); v11 (SEQ ID NO:15); v12 (SEQ IDNO:16); v13 (SEQ ID NO:17); v14 (SEQ ID NO:18); v15 (SEQ ID NO:19); v16(SEQ ID NO:20); v17 (SEQ ID NO:21); v18 (SEQ ID NO:22); v19 (SEQ IDNO:23); v20 (SEQ ID NO:24); v21 (SEQ ID NO:25); v22 (SEQ ID NO:26); v23(SEQ ID NO:27).

FIG. 3 depicts the level of systemic serum exposure of GDF15 in DIO mice(n=7) two weeks following genetic exposure via AAV. As noted in thefigure, for Low AAV the dose=0.7 ng/mL (circles), for Medium AAV thedose=14.9 ng/mL (squares), and for High AAV the dose=65.5 ng/mL(triangles). The horizontal line associated with each dose indicates theaverage.

FIG. 4 depicts the effect on weight reduction in DIO mice prior to(unshaded bars) and following two weeks (shaded bars) of systemicdelivery of GDF15. For AAV dosing, Low=0.7 ng/mL, Medium=14.9 ng/mL, andHigh=65.5 ng/mL. In each group of mice, n=7 and p-values (*, p<0.05; **,p<0.01; ***, p<0.001) were determined by 2-way ANOVA comparing bodyweight at week 2 following GDF15 dosing and PBS vehicle-control dosing.

FIG. 5 depicts the effect on blood glucose, measured after four hours offasting, of DIO mice prior to (unshaded bars) and following two weeks(shaded bars) of systemic delivery of GDF15. For AAV dosing, Low=0.7ng/mL, Medium=14.9 ng/mL, and High=65.5 ng/mL. In each group of mice,n=7 and p-values (*, p<0.05; **, p<0.01; ***, p<0.001) were determinedby 1-way ANOVA comparing blood glucose at week 2 following GDF15 dosingand PBS vehicle-control dosing.

FIG. 6 depicts the effect of GDF15 muteins (v1, v2, v3, v4, v5, v6 andv7; see FIG. 2) on body weight reduction in DIO mice prior to (unshadedbars) and following two weeks (shaded bars) of systemic delivery ofGDF15 at Medium (14.9 ng/mL) AAV dosing. In each group of mice, n=5 andp-values were determined by 1-way ANOVA comparing body weight at week 2following GDF15 mutein dosing and PBS vehicle-control dosing; ns=notsignificant.

FIG. 7 depicts the effect of GDF15 muteins (v1, v2, v3, v4, v5, v6 andv7; see FIG. 2) on fasted serum blood glucose reduction in DIO miceprior to (unshaded bars) and following two weeks (shaded bars) ofsystemic delivery of GDF15 at Medium (14.9 ng/mL) AAV dosing. In eachgroup of mice, n=5 and p-values (*, p<0.05; **, p<0.01; ***, p<0.001)were determined by 1-way ANOVA comparing four-hour fasted serum bloodglucose at week 2 following GDF15 dosing and PBS vehicle-control dosing;ns=not significant.

FIG. 8 depicts the dose-dependent serum exposure of recombinant GDF15,administered twice daily s.c., in 8-week old ob/ob mice (three dosinggroups, n=7 per dosing group) on day 1 and day 7. On day one, the firstdosing group received (w/w equivalent) 0.02 mg/kg, the second dosinggroup received 0.2 mg/kg, and the third dosing group received 2 mg/kg;the serum exposure (measured four hours after administration of the last(second) daily dose) is represented by the unshaded bar (first dosinggroup), grey shaded bar (second dosing group), and black shaded bar(third dosing group). From day 2 to day 7, the first dosing groupreceived (w/w equivalent) 0.01 mg/kg twice daily, the second dosinggroup received 0.1 mg/kg twice daily, and the third dosing groupreceived 1 mg/kg twice daily; the serum exposure (measured four hoursafter administration of the last (second) dose on day 7) is representedby the unshaded bar (first dosing group), grey shaded bar (second dosinggroup), and black shaded bar (third dosing group).

FIG. 9 depicts the dose-dependent effect of recombinant GDF15 on foodintake (grams/day/animal) in ob/ob mice during the course of one week oftwice-daily s.c. dosing. On day one, singly housed animals were fastedin the morning and the first dosing group received (w/w equivalent) 0.02mg/kg, the second dosing group received 0.2 mg/kg, and the third dosinggroup received 2 mg/kg. Eight hours later, this dosing regimen wasrepeated and, thereafter, food was returned to the animal cages. On themorning of day 2, leftover food was weighed and each animal's overnight(0/N) food intake was calculated. For day 2 to day 7, the first dosinggroup received (w/w equivalent) 0.01 mg/kg, the second dosing groupreceived 0.1 mg/kg and the third dosing group received 1 mg/kg. Eachanimal's accumulated food intake from day 2 to day 4 and from day 4 today 7 were again measured. Food intake in vehicle control animals isrepresented by the unshaded bars, while the effect on food intake on thefirst dosing group is represented by the light grey shaded bars, on thesecond dosing group is represented by the medium grey shaded bars, andon the third dosing group is represented by the black shaded bars. Ineach dosing group, n=7 and p-values (*, p<0.05; **, p<0.01; ***,p<0.001) were determined by 2-way ANOVA comparing food intake at eachtime point with vehicle control.

FIG. 10 depicts the dose-dependent effect of twice daily s.c. dosing ofrecombinant GDF15 on body weight (grams) in ob/ob mice at day 1, day 4and day 7. On day one, the first dosing group received (w/w equivalent)0.02 mg/kg, the second dosing group received 0.2 mg/kg, and the thirddosing group received 2 mg/kg. For day 2 to day 7, the first dosinggroup received (w/w equivalent) 0.01 mg/kg, the second dosing groupreceived 0.1 mg/kg and the third dosing group received 1 mg/kg. On eachday, body weight was recorded prior to the morning dose. Body weight invehicle control animals is represented by the unshaded bars, while theeffect of GDF15 on body weight in the first dosing group is representedby the light grey shaded bars, in the second dosing group is representedby the medium grey shaded bars, and in the third dosing group isrepresented by the black shaded bars. In each dosing group, n=7 andp-values (*, p<0.05; **, p<0.01; ***, p<0.001) were determined by 2-wayANOVA comparing food intake at each time point with vehicle control.

FIG. 11 depicts the dose-dependent effect of recombinant GDF15,administered s.c. twice daily, on non-fasted blood glucose measured 16hours post-dose in ob/ob mice. On day one, mice were injected withvehicle (shaded circles), 0.02 mg/kg (first dosing group; shadedsquares), 0.2 mg/kg (second dosing group; shaded triangles) or 2 mg/kg(third dosing group; unshaded circles). For day 2 to day 7, the firstdosing group received (w/w equivalent) 0.01 mg/kg, the second dosinggroup received 0.1 mg/kg and the third dosing group received 1 mg/kg. Atdays 4 and 7, the second and third dosing groups exhibited significantlydecreased non-fasted blood glucose serum levels (mg/dl) relative tovehicle control-injected levels. In each group of mice, n=7 and p-values(*, p<0.05; **, p<0.01; ***, p<0.001) were determined by 2-way ANOVAcomparing non-fasted blood glucose at each time point with vehiclecontrol.

FIG. 12 depicts the effect of recombinant, site-specifically PEGylatedGDF15 muteins on food intake compared to that of the correspondingnon-PEGylated GDF15 muteins in ob/ob mice. Mice received a single s.c.dose of vehicle; non-PEGylated (2 mg/kg) v1, v5 or v6; or PEGylated (2mg/kg) v1, v5 or v6 (see FIG. 2). For day 1, food intake(grams/day/animal) was measured 16 hours following dosing, and then ondays 2, 5-7 and 15. In each group of mice, n=5, and p-values weredetermined by one-way ANOVA comparing food intake at each time pointwith PBS vehicle control.

FIG. 13 depicts the effect of recombinant, site-specifically PEGylatedGDF15 muteins on body weight compared to that of the correspondingnon-PEGylated GDF15 muteins in ob/ob mice. Mice received a single s.c.dose of vehicle; non-PEGylated (2 mg/kg) v1, v5 or v6; or PEGylated (2mg/kg) v1, v5 or v6 (see FIG. 2). For day 1, body weight was measured 16hours following dosing, and then on days 2, 8 and 15. In each group ofmice, n=5, and p-values were determined by one-way ANOVA comparing foodintake at each time point with PBS vehicle control.

DETAILED DESCRIPTION

Before the present disclosure is further described, it is to beunderstood that the disclosure is not limited to the particularembodiments set forth herein, and it is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “theHuman Polypeptide” includes reference to one or more Human Polypeptides;reference to “the Polypeptide” includes reference to one or morePolypeptides; and so forth. It is further noted that the claims may bedrafted to exclude any optional element. As such, this statement isintended to serve as antecedent basis for use of such exclusiveterminology such as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

OVERVIEW

The present disclosure contemplates the use of the agents describedherein, and compositions thereof, to treat and/or prevent variousdiseases, disorders and conditions, and/or the symptoms thereof. In someembodiments, the diseases, disorders and conditions, and/or the symptomsthereof, pertain to glucose metabolism disorders, while in otherembodiments they pertain to body weight disorders. By way of example,but not limitation, the agents, and compositions thereof, can be usedfor the treatment and/or prevention of Type 2 diabetes, insulinresistance and diseases, disorders and conditions characterized byinsulin resistance, decreased insulin production, hyperglycemia,metabolic syndrome, or obesity.

In particular embodiments, the agents contemplated by the presentdisclosure are modified human Growth Differentiation Factor 15 (GDF15)molecules, whereas in other embodiments the agents are GDF15 variants(e.g., muteins) or modified GDF15 variants. The modified human GDF15molecules, GDF15 variants (e.g., muteins) and modified GDF15 variantshave sufficient homology to human GDF15 such that they have the abilityto bind the GDF15 receptor(s) and initiate a signal transduction pathwayresulting in, for example, reduced body weight and/or the otherphysiological effects described herein. The present disclosure alsocontemplates nucleic acid molecules encoding the foregoing. As indicatedabove, the modified human GDF15 molecules, the GDF15 variants (e.g.,muteins), and the modified GDF15 variants described henceforward arecollectively referred to as the “Polypeptide(s)”.

Examples of various GDF15 muteins are described hereafter. In someembodiments, one or more GDF15 residues are substituted with anotheramino acid. In other embodiments, one or more GDF15 native lysineresidues are substituted with another amino acid. However, as set forthbelow, K62Q modifications are inactive. Examples of modified GDF15molecules and modified GDF15 muteins are described hereafter.

The present disclosure contemplates modifications to GDF15 and GDF15muteins, including, for example, pegylation and glycosylation. Inparticular embodiments, strategies are employed such that pegylation iseffected only at specific lysine residues (i.e., site-specificpegylation). The modifications may, for example, improve the serumhalf-life of the Polypeptides. Examples of particular modified GDF15molecules and modified GDF15 muteins are described hereafter.

DEFINITIONS

The terms “patient” or “subject” are used interchangeably to refer to ahuman or a non-human animal (e.g., a mammal).

The terms “treat”, “treating”, treatment” and the like refer to a courseof action (such as administering a Polypeptide or a pharmaceuticalcomposition comprising a Polypeptide) initiated after a disease,disorder or condition, or a symptom thereof, has been diagnosed,observed, and the like so as to eliminate, reduce, suppress, mitigate,or ameliorate, either temporarily or permanently, at least one of theunderlying causes of a disease, disorder, or condition afflicting asubject, or at least one of the symptoms associated with a disease,disorder, condition afflicting a subject. Thus, treatment includesinhibiting (i.e., arresting the development or further development ofthe disease, disorder or condition or clinical symptoms associationtherewith) an active disease (e.g., so as to decrease the level ofinsulin and/or glucose in the bloodstream, to increase glucose toleranceso as to minimize fluctuation of glucose levels, and/or so as to protectagainst diseases caused by disruption of glucose homeostasis).

The term “in need of treatment” as used herein refers to a judgment madeby a physician or other caregiver that a subject requires or willbenefit from treatment. This judgment is made based on a variety offactors that are in the realm of the physician's or caregiver'sexpertise.

The terms “prevent”, “preventing”, “prevention” and the like refer to acourse of action (such as administering a Polypeptide or apharmaceutical composition comprising a Polypeptide) initiated in amanner (e.g., prior to the onset of a disease, disorder, condition orsymptom thereof) so as to prevent, suppress, inhibit or reduce, eithertemporarily or permanently, a subject's risk of developing a disease,disorder, condition or the like (as determined by, for example, theabsence of clinical symptoms) or delaying the onset thereof, generallyin the context of a subject predisposed to having a particular disease,disorder or condition. In certain instances, the terms also refer toslowing the progression of the disease, disorder or condition orinhibiting progression thereof to a harmful or otherwise undesiredstate.

The term “in need of prevention” as used herein refers to a judgmentmade by a physician or other caregiver that a subject requires or willbenefit from preventative care. This judgment is made based on a varietyof factors that are in the realm of a physician's or caregiver'sexpertise.

The phrase “therapeutically effective amount” refers to theadministration of an agent to a subject, either alone or as a part of apharmaceutical composition and either in a single dose or as part of aseries of doses, in an amount that is capable of having any detectable,positive effect on any symptom, aspect, or characteristics of a disease,disorder or condition when administered to a patient. Thetherapeutically effective amount can be ascertained by measuringrelevant physiological effects. For example, in the case of ahyperglycemic condition, a lowering or reduction of blood glucose or animprovement in glucose tolerance test can be used to determine whetherthe amount of an agent is effective to treat the hyperglycemiccondition. For example, a therapeutically effective amount is an amountsufficient to reduce or decrease any level (e.g., a baseline level) offasting plasma glucose (FPG), wherein, for example, the amount issufficient to reduce a FPG level greater than 200 mg/dl to less than 200mg/dl, wherein the amount is sufficient to reduce a FPG level between175 mg/dl and 200 mg/dl to less than the starting level, wherein theamount is sufficient to reduce a FPG level between 150 mg/dl and 175mg/dl to less than the starting level, wherein the amount is sufficientto reduce a FPG level between 125 mg/dl and 150 mg/dl to less than thestarting level, and so on (e.g., reducing FPG levels to less than 125mg/dl, to less than 120 mg/dl, to less than 115 mg/dl, to less than 110mg/dl, etc.). In the case of HbAIc levels, the effective amount is anamount sufficient to reduce or decrease levels by more than about 10% to9%, by more than about 9% to 8%, by more than about 8% to 7%, by morethan about 7% to 6%, by more than about 6% to 5%, and so on. Moreparticularly, a reduction or decrease of HbAIc levels by about 0.1%,0.25%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 3%, 4%, 5%,10%, 20%, 30%, 33%, 35%, 40%, 45%, 50%, or more is contemplated by thepresent disclosure. The therapeutically effective amount can be adjustedin connection with the dosing regimen and diagnostic analysis of thesubject's condition and the like.

The phrase “in a sufficient amount to effect a change” means that thereis a detectable difference between a level of an indicator measuredbefore (e.g., a baseline level) and after administration of a particulartherapy. Indicators include any objective parameter (e.g., level ofglucose or insulin) or subjective parameter (e.g., subject's feeling ofwell-being).

The phrase “glucose tolerance”, as used herein, refers to the ability ofa subject to control the level of plasma glucose and/or plasma insulinwhen glucose intake fluctuates. For example, glucose toleranceencompasses the subject's ability to reduce, within about 120 minutes,the level of plasma glucose back to a level determined before the intakeof glucose.

Broadly speaking, the terms “diabetes” and “diabetic” refer to aprogressive disease of carbohydrate metabolism involving inadequateproduction or utilization of insulin, frequently characterized byhyperglycemia and glycosuria. The terms “pre-diabetes” and“pre-diabetic” refer to a state wherein a subject does not have thecharacteristics, symptoms and the like typically observed in diabetes,but does have characteristics, symptoms and the like that, if leftuntreated, may progress to diabetes. The presence of these conditionsmay be determined using, for example, either the fasting plasma glucose(FPG) test or the oral glucose tolerance test (OGTT). Both require asubject to fast for at least 8 hours prior to initiating the test. Inthe FPG test, a subject's blood glucose is measured after the conclusionof the fasting; generally, the subject fasts overnight and the bloodglucose is measured in the morning before the subject eats. A healthysubject would generally have a FPG concentration between about 90 andabout 100 mg/dl, a subject with “pre-diabetes” would generally have aFPG concentration between about 100 and about 125 mg/dl, and a subjectwith “diabetes” would generally have a FPG level above about 126 mg/dl.In the OGTT, a subject's blood glucose is measured after fasting andagain two hours after drinking a glucose-rich beverage. Two hours afterconsumption of the glucose-rich beverage, a healthy subject generallyhas a blood glucose concentration below about 140 mg/dl, a pre-diabeticsubject generally has a blood glucose concentration about 140 to about199 mg/dl, and a diabetic subject generally has a blood glucoseconcentration about 200 mg/dl or above. While the aforementionedglycemic values pertain to human subjects, normoglycemia, moderatehyperglycemia and overt hyperglycemia are scaled differently in murinesubjects. A healthy murine subject after a four-hour fast wouldgenerally have a FPG concentration between about 100 and about 150mg/dl, a murine subject with “pre-diabetes” would generally have a FPGconcentration between about 175 and about 250 mg/dl and a murine subjectwith “diabetes” would generally have a FPG concentration between aboveabout 250 mg/dl.

The term “insulin resistance” as used herein refers to a condition wherea normal amount of insulin is unable to produce a normal physiologicalor molecular response. In some cases, a hyper-physiological amount ofinsulin, either endogenously produced or exogenously administered, isable to overcome the insulin resistance, in whole or in part, andproduce a biologic response.

The term “metabolic syndrome” refers to an associated cluster of traitsthat includes, but is not limited to, hyperinsulinemia, abnormal glucosetolerance, obesity, redistribution of fat to the abdominal or upper bodycompartment, hypertension, dysfibrinolysis, and dyslipidemiacharacterized by high triglycerides, low high density lipoprotein(HDL)-cholesterol, and high small dense low density lipoprotein (LDL)particles. Subjects having metabolic syndrome are at risk fordevelopment of Type 2 diabetes and, for example, atherosclerosis.

The phrase “glucose metabolism disorder” encompasses any disordercharacterized by a clinical symptom or a combination of clinicalsymptoms that is associated with an elevated level of glucose and/or anelevated level of insulin in a subject relative to a healthy individual.Elevated levels of glucose and/or insulin may be manifested in thefollowing diseases, disorders and conditions: hyperglycemia, type IIdiabetes, gestational diabetes, type I diabetes, insulin resistance,impaired glucose tolerance, hyperinsulinemia, impaired glucosemetabolism, pre-diabetes, metabolic disorders (such as metabolicsyndrome, which is also referred to as syndrome X), and obesity, amongothers. The Polypeptides of the present disclosure, and compositionsthereof, can be used, for example, to achieve and/or maintain glucosehomeostasis, e.g., to reduce glucose level in the bloodstream and/or toreduce insulin level to a range found in a healthy subject.

The term “hyperglycemia”, as used herein, refers to a condition in whichan elevated amount of glucose circulates in the blood plasma of asubject relative to a healthy individual. Hyperglycemia can be diagnosedusing methods known in the art, including measurement of fasting bloodglucose levels as described herein.

The term “hyperinsulinemia”, as used herein, refers to a condition inwhich there are elevated levels of circulating insulin when,concomitantly, blood glucose levels are either elevated or normal.Hyperinsulinemia can be caused by insulin resistance which is associatedwith dyslipidemia such as high triglycerides, high cholesterol, highlow-density lipoprotein (LDL) and low high-density lipoprotein (HDL);high uric acids levels; polycystic ovary syndrome; type II diabetes andobesity. Hyperinsulinemia can be diagnosed as having a plasma insulinlevel higher than about 2 μU/mL.

As used herein, the phrase “body weight disorder” refers to conditionsassociated with excessive body weight and/or enhanced appetite. Variousparameters are used to determine whether a subject is overweightcompared to a reference healthy individual, including the subject's age,height, sex and health status. For example, a subject may be consideredoverweight or obese by assessment of the subject's Body Mass Index(BMI), which is calculated by dividing a subject's weight in kilogramsby the subject's height in meters squared. An adult having a BMI in therange of ˜18.5 to ˜24.9 kg/m² is considered to have a normal weight; anadult having a BMI between ˜25 and ˜29.9 kg/m² may be consideredoverweight (pre-obese); an adult having a BMI of ˜30 kg/m² or higher maybe considered obese. Enhanced appetite frequently contributes toexcessive body weight. There are several conditions associated withenhanced appetite, including, for example, night eating syndrome, whichis characterized by morning anorexia and evening polyphagia oftenassociated with insomnia, but which may be related to injury to thehypothalamus.

The term “Activators” refers to agents that, for example, stimulate,increase, activate, facilitate, enhance activation, sensitize orup-regulate the function or activity of one or more Polypeptides. Inaddition, Activators include agents that operate through the samemechanism of action as the Polypeptides (i.e., agents that modulate thesame signaling pathway as the Polypeptides in a manner analogous to thatof the Polypeptides) and are capable of eliciting a biological responsecomparable to (or greater than) that of the Polypeptides. Examples ofActivators include agonists such as small molecule compounds.

The term “Modulators” collectively refers to the Polypeptides and theActivators.

The terms “modulate”, “modulation” and the like refer to the ability ofthe Modulators to increase the function or activity of one or morePolypeptides (or the nucleic acid molecules encoding them), eitherdirectly or indirectly.

The terms “polypeptide,” “peptide,” and “protein”, used interchangeablyherein, refer to a polymeric form of amino acids of any length, whichcan include genetically coded and non-genetically coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified polypeptide backbones. The terms includefusion proteins, including, but not limited to, fusion proteins with aheterologous amino acid sequence, fusion proteins with heterologous andhomologous leader sequences, with or without N-terminus methionineresidues; immunologically tagged proteins; and the like.

It will be appreciated that throughout this disclosure reference is madeto amino acids according to the single letter or three letter codes. Forthe reader's convenience, the single and three letter amino acid codesare provided below:

G Glycine Gly P Proline Pro A Alanine Ala V Valine Val L Leucine Leu IIsoleucine Ile M Methionine Met C Cysteine Cys F Phenylalanine Phe YTyrosine Tyr W Tryptophan Trp H Histidine His K Lysine Lys R ArginineArg Q Glutamine Gln N Asparagine Asn E Glutamic Acid Glu D Aspartic AcidAsp S Serine Ser T Threonine Thr

As used herein, the term “variant” encompasses naturally-occurringvariants (e.g., homologs and allelic variants) andnon-naturally-occurring variants (e.g., muteins). Naturally-occurringvariants include homologs, i.e., nucleic acids and polypeptides thatdiffer in nucleotide or amino acid sequence, respectively, from onespecies to another. Naturally-occurring variants include allelicvariants, i.e., nucleic acids and polypeptides that differ in nucleotideor amino acid sequence, respectively, from one individual to anotherwithin a species. Non-naturally-occurring variants include nucleic acidsand polypeptides that comprise a change in nucleotide or amino acidsequence, respectively, where the change in sequence is artificiallyintroduced, e.g., the change is generated in the laboratory or otherfacility by human intervention (“hand of man”).

The term “native,” in reference to GDF15, refers to biologically active,naturally-occurring GDF15, including biologically active,naturally-occurring GDF15 variants.

The term “muteins” as used herein refers broadly to mutated recombinantproteins, i.e., a polypeptide comprising an artificially introducedchange in amino acid sequence, e.g., a change in amino acid sequencegenerated in the laboratory or other facility by human intervention(“hand of man”). These proteins usually carry single or multiple aminoacid substitutions and are frequently derived from cloned genes thathave been subjected to site-directed or random mutagenesis, or fromcompletely synthetic genes.

As used herein in reference to native human GDF15 or a GDF15 mutein, theterms “modified”, “modification” and the like refer to one or morechanges that enhance a desired property of human GDF15, anaturally-occurring GDF15 variant, or a GDF15 mutein, where the changedoes not alter the primary amino acid sequence of the GDF15.“Modification” includes a covalent chemical modification that does notalter the primary amino acid sequence of the GDF15 polypeptide itself.Such desired properties include, for example, prolonging the circulationhalf-life, increasing the stability, reducing the clearance, alteringthe immunogenicity or allergenicity, and enabling the raising ofparticular antibodies (e.g., by introduction of unique epitopes) for usein detection assays. Changes to human GDF15, a naturally-occurring GDF15variant, or a GDF15 mutein that may be carried out include, but are notlimited to, pegylation (covalent attachment of one or more molecules ofpolyethylene glycol (PEG), or derivatives thereof); glycosylation (e.g.,N-glycosylation), polysialylation and hesylation; albumin fusion;albumin binding through, for example, a conjugated fatty acid chain(acylation); Fc-fusion; and fusion with a PEG mimetic.

The terms “DNA”, “nucleic acid”, “nucleic acid molecule”,“polynucleotide” and the like are used interchangeably herein to referto a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Non-limiting examples of polynucleotides include linear and circularnucleic acids, messenger RNA (mRNA), complementary DNA (cDNA),recombinant polynucleotides, vectors, probes, primers and the like.

The term “Probe” refers to a fragment of DNA or RNA corresponding to agene or sequence of interest, wherein the fragment has been labeledradioactively (e.g., by incorporating 32^(P) or 35^(S)) or with someother detectable molecule, such as biotin, digoxygenin or fluorescein.As stretches of DNA or RNA with complementary sequences will hybridize,a probe can be used to, for example, label viral plaques, bacterialcolonies or bands on a gel that contain the gene of interest. A probecan be cloned DNA or it can be a synthetic DNA strand; the latter can beused to obtain a cDNA or genomic clone from an isolated protein by, forexample, microsequencing a portion of the protein, deducing the nucleicacid sequence encoding the protein, synthesizing an oligonucleotidecarrying that sequence, radiolabeling the sequence and using it as aprobe to screen a cDNA library or a genomic library.

The term “heterologous” refers to two components that are defined bystructures derived from different sources. For example, in the contextof a polypeptide, a “heterologous” polypeptide may include operablylinked amino acid sequences that are derived from different polypeptides(e.g., a first component comprising a recombinant polypeptide and asecond component derived from a native GDF15 polypeptide). Similarly, inthe context of a polynucleotide encoding a chimeric polypeptide, a“heterologous” polynucleotide may include operably linked nucleic acidsequences that can be derived from different genes (e.g., a firstcomponent from a nucleic acid encoding a polypeptide according to anembodiment disclosed herein and a second component from a nucleic acidencoding a carrier polypeptide). Other exemplary “heterologous” nucleicacids include expression constructs in which a nucleic acid comprising acoding sequence is operably linked to a regulatory element (e.g., apromoter) that is from a genetic origin different from that of thecoding sequence (e.g., to provide for expression in a host cell ofinterest, which may be of different genetic origin than the promoter,the coding sequence or both). For example, a T7 promoter operably linkedto a polynucleotide encoding a GDF15 Polypeptide or domain thereof issaid to be a heterologous nucleic acid. In the context of recombinantcells, “heterologous” can refer to the presence of a nucleic acid (orgene product, such as a polypeptide) that is of a different geneticorigin than the host cell in which it is present.

The term “operably linked” refers to linkage between molecules toprovide a desired function. For example, “operably linked” in thecontext of nucleic acids refers to a functional linkage between anucleic acid expression control sequence (such as a promoter, signalsequence, or array of transcription factor binding sites) and a secondpolynucleotide, wherein the expression control sequence affectstranscription and/or translation of the second polynucleotide. In thecontext of a polypeptide, “operably linked” refers to a functionallinkage between amino acid sequences (e.g., of different domains) toprovide for a described activity of the polypeptide.

As used herein in the context of the structure of a polypeptide,“N-terminus” (or “amino terminus”) and “C-terminus” (or “carboxylterminus”) refer to the extreme amino and carboxyl ends of thepolypeptide, respectively, while the terms “N-terminal” and“C-terminal”refer to relative positions in the amino acid sequence ofthe polypeptide toward the N-terminus and the C-terminus, respectively,and can include the residues at the N-terminus and C-terminus,respectively. “Immediately N-terminal” or “immediately C-terminal”refers to a position of a first amino acid residue relative to a secondamino acid residue where the first and second amino acid residues arecovalently bound to provide a contiguous amino acid sequence.

“Derived from”, in the context of an amino acid sequence orpolynucleotide sequence (e.g., an amino acid sequence “derived from” aGDF15 polypeptide), is meant to indicate that the polypeptide or nucleicacid has a sequence that is based on that of a reference polypeptide ornucleic acid (e.g., a naturally occurring GDF15 polypeptide or aGDF15-encoding nucleic acid), and is not meant to be limiting as to thesource or method in which the protein or nucleic acid is made. By way ofexample, the term “derived from” includes homologues or variants ofreference amino acid or DNA sequences.

“Isolated” refers to a polypeptide of interest that, if naturallyoccurring, is in an environment different from that in which it maynaturally occur. “Isolated” is meant to include polypeptides that arewithin samples that are substantially enriched for the polypeptide ofinterest and/or in which the polypeptide of interest is partially orsubstantially purified. Where the polypeptide is not naturallyoccurring, “isolated” indicates the polypeptide has been separated froman environment in which it was made by either synthetic or recombinantmeans.

“Enriched” means that a sample is non-naturally manipulated (e.g., by ascientist or a clinician) so that a polypeptide of interest is presentin a) a greater concentration (e.g., at least 3-fold greater, at least4-fold greater, at least 8-fold greater, at least 64-fold greater, ormore) than the concentration of the polypeptide in the starting sample,such as a biological sample (e.g., a sample in which the polypeptidenaturally occurs or in which it is present after administration), or b)a concentration greater than the environment in which the polypeptidewas made (e.g., as in a bacterial cell).

“Substantially pure” indicates that a component (e.g., a polypeptide)makes up greater than about 50% of the total content of the composition,and typically greater than about 60% of the total polypeptide content.More typically, “substantially pure” refers to compositions in which atleast 75%, at least 85%, at least 90% or more of the total compositionis the component of interest. In some cases, the polypeptide will makeup greater than about 90%, or greater than about 95% of the totalcontent of the composition.

The terms “antibodies” (Abs) and “immunoglobulins” (Igs) refer toglycoproteins having the same structural characteristics. Whileantibodies exhibit binding specificity to a specific antigen,immunoglobulins include both antibodies and other antibody-likemolecules which lack antigen specificity. Antibodies are described indetail hereafter.

The term “monoclonal antibody” refers to an antibody obtained from apopulation of substantially homogeneous antibodies, that is, theindividual antibodies comprising the population are identical except forpossible naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. In contrast to polyclonal antibodypreparations which include different antibodies directed againstdifferent determinants (epitopes), each monoclonal antibody is directedagainst a single determinant on the antigen.

An “isolated” antibody is one which has been separated and/or recoveredfrom contaminant components of its natural environment; such contaminantcomponents are materials which might interfere with diagnostic ortherapeutic uses for the antibody, and may include enzymes, hormones,and other proteinaceous or nonproteinaceous solutes.

Growth Differentiation Factor 15 (GDF15)

GDF15, also known as MIC-1 (macrophage inhibitory cytokine-1), PDF,PLAB, NAG-1, TGF-PL, and PTGFB, is a member of the transforming growthfactor β (TGF-β) super-family. GDF15, which is synthesized as a 62 kDaintracellular precursor protein that is subsequently cleaved by afurin-like protease, is secreted as a 25 kDa disulfide-linked protein.[See, e.g., Fairlie et al., J. Leukoc. Biol 65:2-5 (1999)]. GDF15 mRNAis seen in several tissues, including liver, kidney, pancreas, colon andplacenta, and GDF15 expression in liver can be significantlyup-regulated during injury of organs such as the liver, kidneys, heartand lungs.

The GDF15 precursor is a 308 amino acid polypeptide (NCBI Ref.Seq.NP_(—)004855.2) containing a 29 amino acid signal peptide, a 167amino acid pro-domain, and a mature domain of 112 amino acids which isexcised from the pro-domain by furin-like proteases. A 308-amino acidGDF15 polypeptide is referred to as a “full-length” GDF15 polypeptide; a112-amino acid GDF15 polypeptide (e.g., amino acids 197-308 of the aminoacid sequence depicted in FIG. 1A) is a “mature” GDF15 polypeptide.Unless otherwise indicated, the term “GDF15” refers to the 112 aminoacid mature sequence. In addition, numerical references to particularGDF15 residues refer to the 112 amino acid mature sequence (i.e.,residue 1 is Ala (A), and residue 112 is Ile (I); see FIG. 1B).

The scope of the present disclosure includes GDF15 orthologs, andmodified forms thereof, from other mammalian species, and their use,including mouse (NP_(—)035949), chimpanzee (XP_(—)524157), orangutan(XP_(—)002828972), Rhesus monkey (EHH29815), giant panda(XP_(—)002912774), gibbon (XP_(—)003275874), guinea pig(XP_(—)003465238), ferret (AER98997), cow (NP_(—)001193227), pig(NP_(—)001167527) and dog (XP_(—)541938). The mature form of human GDF15has approximately 67% amino acid identity to the murine ortholog.

In particular embodiments, the agents contemplated by the presentdisclosure are modified human GDF15 molecules, whereas in otherembodiments the agents are GDF15 variants (e.g., muteins) or modifiedGDF15 variants. The modified human GDF15 molecules, GDF15 variants(e.g., muteins) and modified GDF15 variants have sufficient homology tohuman GDF15 such that they have the ability to bind the GDF15receptor(s) and initiate a signal transduction pathway resulting in, forexample, reduced body weight and/or the other physiological effectsdescribed herein. The present disclosure also contemplates nucleic acidmolecules encoding the foregoing. As indicated above, the modified humanGDF15 molecules, the GDF15 variants (e.g., muteins), and the modifiedGDF15 variants are collectively referred to hereafter as the“Polypeptide(s)”. In addition, fragments of the Polypeptides that retainactivity comparable to, or greater than, native GDF15 are contemplatedby the present disclosure.

The GDF15 muteins contemplated herein include one or more substitutionsof native lysine residues (i.e., residues 62, 69, 91 and 107) with anyother amino acid. However, all GDF15 muteins containing K62Q (e.g.,variants v2, v3 and v4; see FIG. 2) are inactive and behave as negativecontrol proteins. In contrast, GDF15 muteins retaining K62 butincorporating any combination of K69Q, K91R and/or K107R (e.g., variantsv1, v5, v6 and v7; see FIG. 2) are active in lowering body weight to alevel comparable to that of wild-type control. In other GDF15 muteins,one or more GDF15 residue is substituted with another amino acid,including, for example, the following substitutions: H18Q, T19S or V20L.Examples of other GDF15 muteins are set forth hereafter. GDF15 muteinsalso include a GDF15 polypeptide that includes a non-naturally-occurringproline at the amino terminus. As an example, the amino acid sequence ofa naturally-occurring mature GDF15 polypeptide can be modified toinclude an N-terminal proline; the resulting mutein can have a length of113 amino acids. As another example, the amino acid sequence of anon-naturally-occurring GDF15 variant can include an N-terminal proline.

Several techniques, described in detail hereafter, may be used to modifynative GDF15 and GDF15 muteins, including pegylation, N-glycosylation,albumin fusion molecules and Fc-fusion molecules. By way of example,human GDF15 or a GDF15 mutein may be modified by covalent attachment ofone or more PEG molecules at the N-terminus alanine residue (i.e., A1)or at one or more lysine residues within the mature polypeptide (i.e.,residues 62, 69, 91 and 107). In particular embodiments, strategies areemployed such that pegylation is effected only at specific lysineresidues (i.e., site-specific modification). Using standard pegylationtechniques, lysine62 is not modified because it is likely buried withinthe hydrophobic core of the protein dimer interface; thus, it is notnecessary to mutate lysine62 in order to prevent its pegylation(moreover, as noted above, muteins comprising K62Q are inactive).Examples of modified GDF15 molecules and modified GDF15 muteins aredescribed hereafter.

Compared to native GDF15, the modified forms of GDF15, the GDF15variants (e.g., muteins), and the modified GDF15 variants may possesscertain desirable properties, including, for example, improved stabilityand extended serum half-life. Furthermore, as compared to native GDF15,the modified forms of GDF15, the GDF15 variants, and the modified GDF15variants may enable the raising of particular antibodies (e.g., byintroduction of unique epitopes) for use in detection assays, providefor ease of protein purification, etc.

In addition to the Polypeptides, the present disclosure contemplatesother GDF15-related agents (i.e., Activators) capable of eliciting abiological response comparable to (or greater than) that of thePolypeptides, and/or agents capable of enhancing the activity of thePolypeptides.

A. GDF15 Muteins, Modified GDF15 and Modified GDF15 Muteins

The present disclosure contemplates GDF15 muteins wherein one or moreamino acid residues of the mature polypeptide are substituted withanother residue. In particular embodiments, one or more lysine residues(i.e., residues 62, 69, 91 and 107) are substituted with any other aminoacid. Whereas GDF15 muteins containing K62Q are inactive and behave asnegative control proteins (e.g., they do not lower body weight at twoweeks post-administration), GDF15 muteins retaining K62 butincorporating any combination of K69Q, K91R and/or K107R are active inlowering body weight to a level comparable to that of wild-type control.In other embodiments, one or more GDF15 residue is substituted withanother amino acid (e.g., H18Q, T19S or V20L). Examples of GDF15 muteinsinclude, but are not limited to, the following (see FIG. 2):

mutein v1) K69Q, K91R, K107R (SEQ ID NO:5);

mutein v2) K62Q, K91R, K107R (SEQ ID NO:6);

mutein v3) K62Q, K69Q, K107R (SEQ ID NO:7);

mutein v4) K62Q, K69Q, K91R (SEQ ID NO:8);

mutein v5) K91R, K107R (SEQ ID NO:9);

mutein v6) K69Q, K107R (SEQ ID NO:10);

mutein v7) K69Q, K91R (SEQ ID NO:11);

mutein v8) H18Q, T19S, V20L, K62Q, K69Q, K91R, K107R (SEQ ID NO:12);

mutein v9) H18Q, T19S, V20L, K62Q, K91R, K107R (SEQ ID NO:13);

mutein v10) H18Q, T19S, V20L, K62Q, K69Q, K107R (SEQ ID NO:14); and

mutein v11) H18Q, T19S, V20L, K62Q, K69Q, K91R (SEQ ID NO:15).

The effect of particular GDF15 muteins on body weight and fasted glucoseserum levels was compared to that of GDF15. To ensure the accuracy ofsuch a comparison, systemic exposure levels of GDF15 in mice were firstdetermined from the genetic methods described in the Experimentalsection (see, e.g., FIG. 3) in order to establish a clear relationshipof observed phenotype (e.g., body weight and glucose levels) and GDF15serum concentrations (see, e.g., FIGS. 4-7). Using these exposure levelsas a benchmark, mice were subsequently administered recombinant GDF15 atdoses that attained approximately equivalent serum level concentrationsto those observed via the genetic methods. As indicated in FIG. 8,exposure to recombinant GDF15 was within the equivalent range (ng/mL) tothat established in FIG. 3; thus, these data established a correlationof equivalency between serum GDF15 levels and body weight and bloodglucose phenotypes for both the genetic and the recombinant GDF15modalities.

Thereafter, the effects of native mature human GDF15 and particularGDF15 muteins on body weight and fasted glucose serum levels wereevaluated. In particular, the effect of GDF15 and GDF15 muteins v1-v7(see FIG. 2) on body weight reduction in mice is set forth in FIG. 6,and the effect of GDF15 and GDF15 muteins v1-v7 on fasted serum bloodglucose reduction in mice is set forth in FIG. 7. As described in theExperimental section, various GDF15 muteins exhibited similar effects tothose observed with GDF15. For comparison purposes, it was establishedthat administration of recombinant GDF15 significantly decreased foodintake (see FIG. 9) and body weight (see FIG. 10) and resulted insignificantly decreased non-fasted blood glucose serum levels (see FIG.11).

As indicated above and as described in more detail below, in order toenhance one or more properties, native GDF15 and GDF15 muteins may bemodified through, for example, pegylation (covalent attachment of one ormore molecules of polyethylene glycol (PEG), or derivatives thereof);glycosylation (e.g., N-glycosylation) and polysialylation; albuminfusion; albumin binding through, for example, a conjugated fatty acidchain (acylation); Fc-fusion; and fusion with a PEG mimetic. Inparticular embodiments, the modifications are introduced in asite-specific manner.

In some embodiments of the present disclosure, human GDF15 or a GDF15mutein is modified by covalent attachment of one or more PEG moleculesat the N-terminus alanine residue (i.e., A1) and/or at one or morelysine residues within the mature polypeptide (i.e., residues 62, 69, 91and 107). In particular embodiments, strategies are employed such thatpegylation occurs only at specific residues. By way of example, aproline residue may be introduced N-terminal to the alanine residue atposition one (i.e., the N-terminus alanine) to prevent pegylation of thealanine's free amine group. In such variants, residue one is theN-terminus proline and residue 113 is isoleucine. Native mature GDF15and GDF15 muteins containing the N-terminus proline include, but are notlimited to, the following (see FIG. 2):

mutein v12) NPro-GDF15 (SEQ ID NO:16);

mutein v13) NPro, K70Q, K92R, K108R (SEQ ID NO:17);

mutein v14) NPro, K63Q, K92R, K108R (SEQ ID NO:18);

mutein v15) NPro, K63Q, K70Q, K108R (SEQ ID NO:19);

mutein v16) NPro, K63Q, K70Q, K92R (SEQ ID NO:20);

mutein v17) NPro, K92R, K108R (SEQ ID NO:21);

mutein v18) NPro, K70Q, K108R (SEQ ID NO:22);

mutein v19) NPro, K70Q, K92R (SEQ ID NO:23);

mutein v20) NPro, H19Q, T205, V21L, K63Q, K70Q, K92R, K108R (SEQ IDNO:24);

mutein v21) NPro, H19Q, T205, V21L, K63Q, K92R, K108R (SEQ ID NO:25);

mutein v22) NPro, H19Q, T205, V21L, K63Q, K70Q, K108R (SEQ ID NO:26);and

mutein v23) NPro, H19Q, T205, V21L, K63Q, K70Q, K92R (SEQ ID NO:27).

Moreover, as detailed in the Experimental section, N-Hydroxysuccinimide(NHS)-specific chemistries may be employed to modify the recombinantGDF15 muteins of the present disclosure in a site-specific manner. Forexample, when desired, prevention of PEGylation at the N-terminalalanine can be achieved using sulfo-NHS-acetate to introduce an acetylmoiety that serves as a protecting group. In order to prevent pegylationof one or more internal lysine residues, lysine substitutions such asthose described above can be introduced; because lysine62 is notpegylated using standard techniques, it is unnecessary to mutate theresidue in order to prevent its pegylation. Using these NHS chemistries,the following variants were modified by attachment of a linear 10 kDaPEG moiety (see Example 4): variant 1 was PEGylated at the N-terminus(v1-PEG10) (SEQ ID NO:5); variant 5 was PEGylated at lysine69 (v5-PEG10)(SEQ ID NO:9); and variant 6 was PEGylated at lysine91 (v6-PEG10) (SEQID NO:10).

Nucleic acid molecules encoding the Polypeptides are contemplated by thepresent disclosure, including their naturally-occurring andnon-naturally occurring isoforms, allelic variants and splice variants.As previously noted, a Polypeptide also refers to polypeptides that haveone or more alterations in the amino acid residues (e.g., at locationsthat are not conserved across variants or species) while retaining theconserved domains and having the same biological activity as thenaturally-occurring Polypeptides. The present disclosure alsoencompasses nucleic acid sequences that vary in one or more bases from anaturally-occurring DNA sequence but still translate into an amino acidsequence that corresponds to a Polypeptide due to degeneracy of thegenetic code. For example, GDF15 may refer to amino acid sequences thatdiffer from the naturally-occurring sequence by one or more conservativesubstitutions, tags, or conjugates (e.g., a Polypeptide).

Thus, in addition to any naturally-occurring GDF15 polypeptide, thepresent disclosure contemplates having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10usually no more than 20, 10, or 5 amino acid substitutions, where thesubstitution is usually a conservative amino acid substitution (e.g., aPolypeptide).

By “conservative amino acid substitution” generally refers tosubstitution of amino acid residues within the following groups: 1) L,I, M, V, F; 2) R, K; 3) F, Y, H, W, R; 4) G, A, T, S; 5) Q, N; and 6) D,E. Conservative amino acid substitutions preserve the activity of theprotein by replacing an amino acid(s) in the protein with an amino acidwith a side chain of similar acidity, basicity, charge, polarity, orsize of the side chain. Guidance for substitutions, insertions, ordeletions may be based on alignments of amino acid sequences ofdifferent variant proteins or proteins from different species.

The present disclosure also contemplates active fragments (e.g.,subsequences) of the Polypeptides containing contiguous amino acidresidues derived from the mature GDF15 polypeptide or a GDF15 mutein.The length of contiguous amino acid residues of a peptide or apolypeptide subsequence varies depending on the specificnaturally-occurring amino acid sequence from which the subsequence isderived. In general, peptides and polypeptides may be from about 5 aminoacids to about 10 amino acids, from about 10 amino acids to about 15amino acids, from about 15 amino acids to about 20 amino acids, fromabout 20 amino acids to about 25 amino acids, from about 25 amino acidsto about 30 amino acids, from about 30 amino acids to about 40 aminoacids, from about 40 amino acids to about 50 amino acids, from about 50amino acids to about 75 amino acids, from about 75 amino acids to about100 amino acids, or from about 100 amino acids up to the length of themature peptide or polypeptide.

Additionally, the Polypeptides can have a defined sequence identitycompared to a reference sequence over a defined length of contiguousamino acids (e.g., a “comparison window”). Methods of alignment ofsequences for comparison are well-known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by thehomology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443(1970), by the search for similarity method of Pearson & Lipman, Proc.Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Madison, Wis.), or by manual alignment andvisual inspection (see, e.g., Current Protocols in Molecular Biology(Ausubel et al., eds. 1995 supplement)).

As an example, a suitable Polypeptide can comprise an amino acidsequence having at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, or atleast about 99%, amino acid sequence identity to a contiguous stretch offrom about 5 amino acids to about 10 amino acids, from about 10 aminoacids to 12 amino acids, from about 12 amino acids to about 15 aminoacids, from about 15 amino acids to about 20 amino acids, from about 20amino acids to about 25 amino acids, from about 25 amino acids to about30 amino acids from about 30 amino acids to about 35 amino acids, fromabout 35 amino acids to about 40 amino acids, from about 40 amino acidsto about 45 amino acids, from about 45 amino acids to about 50 aminoacids, from about 50 amino acids to about 60 amino acids, from about 60amino acids to about 70 amino acids, from about 70 amino acids to about80 amino acids, from about 80 amino acids to about 90 amino acids, fromabout 90 amino acids to about 100 amino acids, or from about 100 aminoacids to 112 amino acids or 113 amino acids, of one of the followingreference amino acid sequences: v1-v23 (see, e.g., FIG. 2).

The Polypeptides may be isolated from a natural source (e.g., anenvironment other than its naturally-occurring environment) and also maybe recombinantly made (e.g., in a genetically modified host cell such asbacteria; yeast; Pichia; insect cells; and the like), where thegenetically modified host cell is modified with a nucleic acidcomprising a nucleotide sequence encoding the polypeptide. ThePolypeptides may also be synthetically produced (e.g., by cell-freechemical synthesis). Methods of productions are described in more detailbelow.

A Polypeptide may be generated using recombinant techniques tomanipulate different GDF15-related nucleic acids known in the art toprovide constructs capable of encoding the Polypeptide. It will beappreciated that, when provided a particular amino acid sequence, theordinary skilled artisan will recognize a variety of different nucleicacid molecules encoding such amino acid sequence in view of herbackground and experience in, for example, molecular biology.

B. Modulators

The term “Modulators” refers to both Polypeptides and Activators. Asindicated above, Activators are agents that, for example, stimulate,increase, activate, facilitate, enhance activation, sensitize orup-regulate the function or activity of one or more Polypeptides. Inaddition, Activators include agents that operate through the samemechanism of action as the Polypeptides (i.e., agents that modulate thesame signaling pathway as the Polypeptides in a manner analogous to thatof the Polypeptides) and are capable of eliciting a biological responsecomparable to (or greater than) that of the Polypeptides. An Activatormay be, for example, a small molecule agonist compound, or otherbioorganic molecule.

In some embodiments, the Activator is a small molecule agonist compound.Numerous libraries of small molecule compounds (e.g., combinatoriallibraries) are commercially available and can serve as a starting pointfor identifying such an Activator. The skilled artisan is able todevelop one or more assays (e.g., biochemical or cell-based assays) inwhich such compound libraries can be screened in order to identify oneor more compounds having the desired properties; thereafter, the skilledmedicinal chemist is able to optimize such one or more compounds by, forexample, synthesizing and evaluating analogs and derivatives thereof.Synthetic and/or molecular modeling studies can also be utilized in theidentification of an Activator.

In still further embodiments, the Activator is an agonistic polypeptidestructurally distinguishable from the Polypeptides but having comparableactivity. The skilled artisan is able to identify such polypeptideshaving desired properties.

Amide Bond Substitutions

In some cases, a Polypeptide includes one or more linkages other thanpeptide bonds, e.g., at least two adjacent amino acids are joined via alinkage other than an amide bond. For example, in order to reduce oreliminate undesired proteolysis or other means of degradation, and/or toincrease serum stability, and/or to restrict or increase conformationalflexibility, one or more amide bonds within the backbone of aPolypeptide can be substituted.

In another example, one or more amide linkages (—CO—NH—) in aPolypeptide can be replaced with a linkage which is an isostere of anamide linkage, such as —CH₂NH—, CH₂S—, —CH₂CH₂—, —CH═CH-(cis and trans),—COCH₂—, —CH(OH)CH₂— or —CH₂SO—. One or more amide linkages in aPolypeptide can also be replaced by, for example, a reduced isosterepseudopeptide bond. See Couder et al. (1993) Int. J. Peptide ProteinRes. 41:181-184. Such replacements and how to effect are known to thoseof ordinary skill in the art.

Amino Acid Substitutions

One or more amino acid substitutions can be made in a Polypeptide. Thefollowing are non-limiting examples:

a) substitution of alkyl-substituted hydrophobic amino acids, includingalanine, leucine, isoleucine, valine, norleucine, (S)-2-aminobutyricacid, (S)-cyclohexylalanine or other simple alpha-amino acidssubstituted by an aliphatic side chain from C₁-C₁₀ carbons includingbranched, cyclic and straight chain alkyl, alkenyl or alkynylsubstitutions;

b) substitution of aromatic-substituted hydrophobic amino acids,including phenylalanine, tryptophan, tyrosine, sulfotyrosine,biphenylalanine, 1-naphthylalanine, 2-naphthylalanine,2-benzothienylalanine, 3-benzothienylalanine, histidine, includingamino, alkylamino, dialkylamino, aza, halogenated (fluoro, chloro,bromo, or iodo) or alkoxy (from C₁-C₄)-substituted forms of theabove-listed aromatic amino acids, illustrative examples of which are:2-, 3- or 4-aminophenylalanine, 2-, 3- or 4-chlorophenylalanine, 2-, 3-or 4-methylphenylalanine, 2-, 3- or 4-methoxyphenylalanine, 5-amino-,5-chloro-, 5-methyl- or 5-methoxytryptophan, 2′-, 3′-, or 4′-amino-,2′-, 3′-, or 4′-chloro-, 2, 3, or 4-biphenylalanine, 2′-, 3′-, or4′-methyl-, 2-, 3- or 4-biphenylalanine, and 2- or 3-pyridylalanine;

c) substitution of amino acids containing basic side chains, includingarginine, lysine, histidine, ornithine, 2,3-diaminopropionic acid,homoarginine, including alkyl, alkenyl, or aryl-substituted (from C₁-C₁₀branched, linear, or cyclic) derivatives of the previous amino acids,whether the substituent is on the heteroatoms (such as the alphanitrogen, or the distal nitrogen or nitrogens, or on the alpha carbon,in the pro-R position for example. Compounds that serve as illustrativeexamples include: N-epsilon-isopropyl-lysine,3-(4-tetrahydropyridyl)-glycine, 3-(4-tetrahydropyridyl)-alanine,N,N-gamma, gamma′-diethyl-homoarginine. Included also are compounds suchas alpha-methyl-arginine, alpha-methyl-2,3-diaminopropionic acid,alpha-methyl-histidine, alpha-methyl-ornithine where the alkyl groupoccupies the pro-R position of the alpha-carbon. Also included are theamides formed from alkyl, aromatic, heteroaromatic (where theheteroaromatic group has one or more nitrogens, oxygens or sulfur atomssingly or in combination) carboxylic acids or any of the many well-knownactivated derivatives such as acid chlorides, active esters, activeazolides and related derivatives) and lysine, ornithine, or2,3-diaminopropionic acid;

d) substitution of acidic amino acids, including aspartic acid, glutamicacid, homoglutamic acid, tyrosine, alkyl, aryl, arylalkyl, andheteroaryl sulfonamides of 2,4-diaminopriopionic acid, ornithine orlysine and tetrazole-substituted alkyl amino acids;

e) substitution of side chain amide residue, including asparagine,glutamine, and alkyl or aromatic substituted derivatives of asparagineor glutamine; and

f) substitution of hydroxyl containing amino acids, including serine,threonine, homoserine, 2,3-diaminopropionic acid, and alkyl or aromaticsubstituted derivatives of serine or threonine.

In some cases, a Polypeptide comprises one or more naturally occurringnon-genetically encoded L-amino acids, synthetic L-amino acids orD-enantiomers of an amino acid. For example, a Polypeptide can compriseonly D-amino acids. For example, a Polypeptide can comprise one or moreof the following residues: hydroxyproline, β-alanine, o-aminobenzoicacid, m-aminobenzoic acid, p-aminobenzoic acid, m-aminomethylbenzoicacid, 2,3-diaminopropionic acid, α-aminoisobutyric acid, N-methylglycine(sarcosine), ornithine, citrulline, t-butylalanine, t-butylglycine,N-methylisoleucine, phenylglycine, cyclohexylalanine, norleucine,naphthylalanine, pyridylalanine 3-benzothienyl alanine,4-chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine,4-fluorophenylalanine, penicillamine,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, β-2-thienylalanine,methionine sulfoxide, homoarginine, N-acetyl lysine, 2,4-diamino butyricacid, rho-aminophenylalanine, N-methylvaline, homocysteine, homoserine,ε-amino hexanoic acid, ω-aminohexanoic acid, ω-aminoheptanoic acid,ω-aminooctanoic acid, ω-aminodecanoic acid, ω-aminotetradecanoic acid,cyclohexylalanine, α,γ-diaminobutyric acid, α,β-diaminopropionic acid,δ-amino valeric acid, and 2,3-diaminobutyric acid.

Additional Modifications

A cysteine residue or a cysteine analog can be introduced into aPolypeptide to provide for linkage to another peptide via a disulfidelinkage or to provide for cyclization of the Polypeptide. Methods ofintroducing a cysteine or cysteine analog are known in the art; see,e.g., U.S. Pat. No. 8,067,532.

A Polypeptide can be cyclized. One or more cysteine or cysteine analogscan be introduced into a Polypeptide, where the introduced cysteine orcysteine analog can form a disulfide bond with a second introducedcysteine or cysteine analog. Other means of cyclization includeintroduction of an oxime linker or a lanthionine linker; see, e.g., U.S.Pat. No. 8,044,175. Any combination of amino acids (or non-amino acidmoiety) that can form a cyclizing bond can be used and/or introduced. Acyclizing bond can be generated with any combination of amino acids (orwith amino acid and —(CH₂)_(n)—CO— or —(CH₂)_(n)—C₆H₄—CO—) withfunctional groups which allow for the introduction of a bridge. Someexamples are disulfides, disulfide mimetics such as the —(CH₂)_(n)—carba bridge, thioacetal, thioether bridges (cystathionine orlanthionine) and bridges containing esters and ethers. In theseexamples, n can be any integer, but is frequently less than ten.

Other modifications include, for example, an N-alkyl (or aryl)substitution (ψ[CONR]), or backbone crosslinking to construct lactamsand other cyclic structures. Other derivatives of the modulatorcompounds of the present disclosure include C-terminal hydroxymethylderivatives, O-modified derivatives (e.g., C-terminal hydroxymethylbenzyl ether), N-terminally modified derivatives including substitutedamides such as alkylamides and hydrazides.

In some cases, one or more L-amino acids in a Polypeptide is replacedwith a D-amino acid.

In some cases, a Polypeptide is a retroinverso analog. Sela and Zisman(1997) FASEB J. 11:449. Retro-inverso peptide analogs are isomers oflinear polypeptides in which the direction of the amino acid sequence isreversed (retro) and the chirality, D- or L-, of one or more amino acidstherein is inverted (inverso) e.g., using D-amino acids rather thanL-amino acids. See, e.g., Jameson et al. (1994) Nature 368:744; andBrady et al. (1994) Nature 368:692.

A Polypeptide can include a “Protein Transduction Domain” (PTD), whichrefers to a polypeptide, polynucleotide, carbohydrate, or organic orinorganic compound that facilitates traversing a lipid bilayer, micelle,cell membrane, organelle membrane, or vesicle membrane. A PTD attachedto another molecule facilitates the molecule traversing a membrane, forexample going from extracellular space to intracellular space, orcytosol to within an organelle. In some embodiments, a PTD is covalentlylinked to the amino terminus of a Polypeptide, while in otherembodiments, a PTD is covalently linked to the carboxyl terminus of aPolypeptide. Exemplary protein transduction domains include, but are notlimited to, a minimal undecapeptide protein transduction domain(corresponding to residues 47-57 of HIV-1 TAT comprising YGRKKRRQRRR;SEQ ID NO:28); a polyarginine sequence comprising a number of argininessufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10,or 10-50 arginines); a VP22 domain (Zender et al. (2002) Cancer GeneTher. 9(6):489-96); an Drosophila Antennapedia protein transductiondomain (Noguchi et al. (2003) Diabetes 52(7):1732-1737); a truncatedhuman calcitonin peptide (Trehin et al. (2004) Pharm. Research21:1248-1256); polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci.USA 97:13003-13008); RRQRRTSKLMKR (SEQ ID NO:29); TransportanGWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:30);KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:31); and RQIKIWFQNRRMKWKK(SEQ ID NO:32). Exemplary PTDs include, but are not limited to,YGRKKRRQRRR (SEQ ID NO:28), RKKRRQRRR (SEQ ID NO:33); an argininehomopolymer of from 3 arginine residues to 50 arginine residues;Exemplary PTD domain amino acid sequences include, but are not limitedto, any of the following: YGRKKRRQRRR (SEQ ID NO:28); RKKRRQRR (SEQ IDNO:34); YARAAARQARA (SEQ ID NO:35); THRLPRRRRRR (SEQ ID NO:36); andGGRRARRRRRR (SEQ ID NO:37).

The carboxyl group COR₃ of the amino acid at the C-terminal end of aPolypeptide can be present in a free form (R₃═OH) or in the form of aphysiologically-tolerated alkaline or alkaline earth salt such as, e.g.,a sodium, potassium or calcium salt. The carboxyl group can also beesterified with primary, secondary or tertiary alcohols such as, e.g.,methanol, branched or unbranched C₁-C₆-alkyl alcohols, e.g., ethylalcohol or tert-butanol. The carboxyl group can also be amidated withprimary or secondary amines such as ammonia, branched or unbranchedC₁-C₆-alkylamines or C₁-C₆ di-alkylamines, e.g., methylamine ordimethylamine.

The amino group of the amino acid NR₁R₂ at the N-terminus of aPolypeptide can be present in a free form (R₁═H and R₂═H) or in the formof a physiologically-tolerated salt such as, e.g., a chloride oracetate. The amino group can also be acetylated with acids such thatR₁═H and R₂=acetyl, trifluoroacetyl, or adamantyl. The amino group canbe present in a form protected by amino-protecting groups conventionallyused in peptide chemistry such as, e.g., Fmoc, Benzyloxy-carbonyl (Z),Boc, or Alloc. The amino group can be N-alkylated in which R₁ and/orR₂═C₁-C₆ alkyl or C₂-C₈ alkenyl or C₇-C₉ aralkyl. Alkyl residues can bestraight-chained, branched or cyclic (e.g., ethyl, isopropyl andcyclohexyl, respectively).

Particular Modifications to Enhance and/or Mimic GDF15 Function

A Polypeptide can include one or more modifications that enhance aproperty desirable in a protein formulated for therapy (e.g., serumhalf-life), that enable the raising of antibodies for use in detectionassays (e.g., epitope tags), that provide for ease of proteinpurification, etc. Such modifications include, but are not limited to,including pegylation (covalent attachment of one or more molecules ofpolyethylene glycol (PEG), or derivatives thereof); N-glycosylation andpolysialylation; albumin fusion; albumin binding through a conjugatedfatty acid chain (acylation); Fc-fusion proteins; and fusion with a PEGmimetic.

Pegylation:

The clinical effectiveness of protein therapeutics is often limited byshort plasma half-life and susceptibility to protease degradation.Studies of various therapeutic proteins (e.g., filgrastim) have shownthat such difficulties may be overcome by various modifications,including conjugating or linking the polypeptide sequence to any of avariety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG),polypropylene glycol, or polyoxyalkylenes (see, for example, typicallyvia a linking moiety covalently bound to both the protein and thenonproteinaceous polymer, e.g., a PEG). Such PEG-conjugated biomoleculeshave been shown to possess clinically useful properties, includingbetter physical and thermal stability, protection against susceptibilityto enzymatic degradation, increased solubility, longer in vivocirculating half-life and decreased clearance, reduced immunogenicityand antigenicity, and reduced toxicity.

PEGs suitable for conjugation to a polypeptide sequence are generallysoluble in water at room temperature, and have the general formulaR(O—CH₂—CH₂)_(n)O—R, where R is hydrogen or a protective group such asan alkyl or an alkanol group, and where n is an integer from 1 to 1000.When R is a protective group, it generally has from 1 to 8 carbons. ThePEG conjugated to the polypeptide sequence can be linear or branched.Branched PEG derivatives, “star-PEGs” and multi-armed PEGs arecontemplated by the present disclosure. A molecular weight of the PEGused in the present disclosure is not restricted to any particularrange, but certain embodiments have a molecular weight between 500 and20,000 while other embodiments have a molecular weight between 4,000 and10,000.

The present disclosure also contemplates compositions of conjugateswherein the PEGs have different n values and thus the various differentPEGs are present in specific ratios. For example, some compositionscomprise a mixture of conjugates where n=1, 2, 3 and 4. In somecompositions, the percentage of conjugates where n=1 is 18-25%, thepercentage of conjugates where n=2 is 50-66%, the percentage ofconjugates where n=3 is 12-16%, and the percentage of conjugates wheren=4 is up to 5%. Such compositions can be produced by reactionconditions and purification methods know in the art. For example, cationexchange chromatography may be used to separate conjugates, and afraction is then identified which contains the conjugate having, forexample, the desired number of PEGs attached, purified free fromunmodified protein sequences and from conjugates having other numbers ofPEGs attached.

PEG may be bound to a polypeptide of the present disclosure via aterminal reactive group (a “spacer”). The spacer is, for example, aterminal reactive group which mediates a bond between the free amino orcarboxyl groups of one or more of the polypeptide sequences andpolyethylene glycol. The PEG having the spacer which may be bound to thefree amino group includes N-hydroxysuccinylimide polyethylene glycolwhich may be prepared by activating succinic acid ester of polyethyleneglycol with N-hydroxysuccinylimide. Another activated polyethyleneglycol which may be bound to a free amino group is2,4-bis(O-methoxypolyethyleneglycol)-6-chloro-s-triazine which may beprepared by reacting polyethylene glycol monomethyl ether with cyanuricchloride. The activated polyethylene glycol which is bound to the freecarboxyl group includes polyoxyethylenediamine.

Conjugation of one or more of the polypeptide sequences of the presentdisclosure to PEG having a spacer may be carried out by variousconventional methods. For example, the conjugation reaction can becarried out in solution at a pH of from 5 to 10, at temperature from 4°C. to room temperature, for 30 minutes to 20 hours, utilizing a molarratio of reagent to protein of from 4:1 to 30:1. Reaction conditions maybe selected to direct the reaction towards producing predominantly adesired degree of substitution. In general, low temperature, low pH(e.g., pH=5), and short reaction time tend to decrease the number ofPEGs attached, whereas high temperature, neutral to high pH (e.g.,pH≧7), and longer reaction time tend to increase the number of PEGsattached. Various means known in the art may be used to terminate thereaction. In some embodiments the reaction is terminated by acidifyingthe reaction mixture and freezing at, e.g., −20° C.

The present disclosure also contemplates the use of PEG Mimetics.Recombinant PEG mimetics have been developed that retain the attributesof PEG (e.g., enhanced serum half-life) while conferring severaladditional advantageous properties. By way of example, simplepolypeptide chains (comprising, for example, Ala, Glu, Gly, Pro, Ser andThr) capable of forming an extended conformation similar to PEG can beproduced recombinantly already fused to the peptide or protein drug ofinterest (e.g., Amunix′ XTEN technology; Mountain View, Calif.). Thisobviates the need for an additional conjugation step during themanufacturing process. Moreover, established molecular biologytechniques enable control of the side chain composition of thepolypeptide chains, allowing optimization of immunogenicity andmanufacturing properties.

Glycosylation and Polysialylation:

For purposes of the present disclosure, “glycosylation” is meant tobroadly refer to the enzymatic process that attaches glycans toproteins, lipids or other organic molecules. The use of the term“glycosylation” in conjunction with the present disclosure is generallyintended to mean adding or deleting one or more carbohydrate moieties(either by removing the underlying glycosylation site or by deleting theglycosylation by chemical and/or enzymatic means), and/or adding one ormore glycosylation sites that may or may not be present in the nativesequence. In addition, the phrase includes qualitative changes in theglycosylation of the native proteins involving a change in the natureand proportions of the various carbohydrate moieties present.

Glycosylation can dramatically affect the physical properties ofproteins and can also be important in protein stability, secretion, andsubcellular localization. Proper glycosylation can be essential forbiological activity. In fact, some genes from eucaryotic organisms, whenexpressed in bacteria (e.g., E. coli) which lack cellular processes forglycosylating proteins, yield proteins that are recovered with little orno activity by virtue of their lack of glycosylation.

Addition of glycosylation sites can be accomplished by altering theamino acid sequence. The alteration to the polypeptide may be made, forexample, by the addition of, or substitution by, one or more serine orthreonine residues (for O-linked glycosylation sites) or asparagineresidues (for N-linked glycosylation sites). The structures of N-linkedand O-linked oligosaccharides and the sugar residues found in each typemay be different. One type of sugar that is commonly found on both isN-acetylneuraminic acid (hereafter referred to as sialic acid). Sialicacid is usually the terminal residue of both N-linked and O-linkedoligosaccharides and, by virtue of its negative charge, may conferacidic properties to the glycoprotein. A particular embodiment of thepresent disclosure comprises the generation and use of N-glycosylationvariants.

The polypeptide sequences of the present disclosure may optionally bealtered through changes at the DNA level, particularly by mutating theDNA encoding the polypeptide at preselected bases such that codons aregenerated that will translate into the desired amino acids. Anothermeans of increasing the number of carbohydrate moieties on thepolypeptide is by chemical or enzymatic coupling of glycosides to thepolypeptide. Removal of carbohydrates may be accomplished chemically orenzymatically, or by substitution of codons encoding amino acid residuesthat are glycosylated. Chemical deglycosylation techniques are known,and enzymatic cleavage of carbohydrate moieties on polypeptides can beachieved by the use of a variety of endo- and exo-glycosidases.

Dihydrofolate reductase (DHFR)-deficient Chinese Hamster Ovary (CHO)cells are a commonly used host cell for the production of recombinantglycoproteins. These cells do not express the enzyme beta-galactosidealpha-2,6-sialyltransferase and therefore do not add sialic acid in thealpha-2,6 linkage to N-linked oligosaccharides of glycoproteins producedin these cells.

The present disclosure also contemplates the use of polysialylation, theconjugation of peptides and proteins to the naturally occurring,biodegradable α-(2→8) linked polysialic acid (“PSA”) in order to improvetheir stability and in vivo pharmacokinetics. PSA is a biodegradable,non-toxic natural polymer that is highly hydrophilic, giving it a highapparent molecular weight in the blood which increases its serumhalf-life. In addition, polysialylation of a range of peptide andprotein therapeutics has led to markedly reduced proteolysis, retentionof activity in vivo activity, and reduction in immunogenicity andantigenicity (see, e.g., G. Gregoriadis et al., Int. J. Pharmaceutics300(1-2):125-30). As with modifications with other conjugates (e.g.,PEG), various techniques for site-specific polysialylation are available(see, e.g., T. Lindhout et al., PNAS 108(18)7397-7402 (2011)).

Albumin Fusion and Conjugation with Other Molecules:

Additional suitable components and molecules for conjugation include,for example, thyroglobulin; albumins such as human serum albumin (HAS);tetanus toxoid; Diphtheria toxoid; polyamino acids such aspoly(D-lysine:D-glutamic acid); VP6 polypeptides of rotaviruses;influenza virus hemaglutinin, influenza virus nucleoprotein; KeyholeLimpet Hemocyanin (KLH); and hepatitis B virus core protein and surfaceantigen; or any combination of the foregoing.

Fusion of albumin to one or more polypeptides of the present disclosurecan, for example, be achieved by genetic manipulation, such that the DNAcoding for HSA, or a fragment thereof, is joined to the DNA coding forthe one or more polypeptide sequences. Thereafter, a suitable host canbe transformed or transfected with the fused nucleotide sequences in theform of, for example, a suitable plasmid, so as to express a fusionpolypeptide. The expression may be effected in vitro from, for example,prokaryotic or eukaryotic cells, or in vivo from, for example, atransgenic organism. In some embodiments of the present disclosure, theexpression of the fusion protein is performed in mammalian cell lines,for example, CHO cell lines. Transformation is used broadly herein torefer to the genetic alteration of a cell resulting from the directuptake, incorporation and expression of exogenous genetic material(exogenous DNA) from its surroundings and taken up through the cellmembrane(s). Transformation occurs naturally in some species ofbacteria, but it can also be effected by artificial means in othercells.

Furthermore, albumin itself may be modified to extend its circulatinghalf-life. Fusion of the modified albumin to one or more Polypeptidescan be attained by the genetic manipulation techniques described aboveor by chemical conjugation; the resulting fusion molecule has ahalf-life that exceeds that of fusions with non-modified albumin. [SeeWO2011/051489].

Several albumin-binding strategies have been developed as alternativesfor direct fusion, including albumin binding through a conjugated fattyacid chain (acylation). Because serum albumin is a transport protein forfatty acids, these natural ligands with albumin-binding activity havebeen used for half-life extension of small protein therapeutics. Forexample, insulin determir (LEVEMIR), an approved product for diabetes,comprises a myristyl chain conjugated to a genetically-modified insulin,resulting in a long-acting insulin analog.

Another type of modification is to conjugate (e.g., link) one or moreadditional components or molecules at the N- and/or C-terminus of apolypeptide sequence, such as another protein (e.g., a protein having anamino acid sequence heterologous to the subject protein), or a carriermolecule. Thus, an exemplary polypeptide sequence can be provided as aconjugate with another component or molecule.

A conjugate modification may result in a polypeptide sequence thatretains activity with an additional or complementary function oractivity of the second molecule. For example, a polypeptide sequence maybe conjugated to a molecule, e.g., to facilitate solubility, storage, invivo or shelf half-life or stability, reduction in immunogenicity,delayed or controlled release in vivo, etc. Other functions oractivities include a conjugate that reduces toxicity relative to anunconjugated polypeptide sequence, a conjugate that targets a type ofcell or organ more efficiently than an unconjugated polypeptidesequence, or a drug to further counter the causes or effects associatedwith a disorder or disease as set forth herein (e.g., diabetes).

A Polypeptide may also be conjugated to large, slowly metabolizedmacromolecules such as proteins; polysaccharides, such as sepharose,agarose, cellulose, cellulose beads; polymeric amino acids such aspolyglutamic acid, polylysine; amino acid copolymers; inactivated virusparticles; inactivated bacterial toxins such as toxoid from diphtheria,tetanus, cholera, leukotoxin molecules; inactivated bacteria; anddendritic cells. Such conjugated forms, if desired, can be used toproduce antibodies against a polypeptide of the present disclosure.

Additional candidate components and molecules for conjugation includethose suitable for isolation or purification. Particular non-limitingexamples include binding molecules, such as biotin (biotin-avidinspecific binding pair), an antibody, a receptor, a ligand, a lectin, ormolecules that comprise a solid support, including, for example, plasticor polystyrene beads, plates or beads, magnetic beads, test strips, andmembranes.

Purification methods such as cation exchange chromatography may be usedto separate conjugates by charge difference, which effectively separatesconjugates into their various molecular weights. For example, the cationexchange column can be loaded and then washed with ˜20 mM sodiumacetate, pH˜4, and then eluted with a linear (0 M to 0.5 M) NaClgradient buffered at a pH from about 3 to 5.5, e.g., at pH˜4.5. Thecontent of the fractions obtained by cation exchange chromatography maybe identified by molecular weight using conventional methods, forexample, mass spectroscopy, SDS-PAGE, or other known methods forseparating molecular entities by molecular weight.

Fc-Fusion Molecules:

In certain embodiments, the amino- or carboxyl-terminus of a polypeptidesequence of the present disclosure can be fused with an immunoglobulinFc region (e.g., human Fc) to form a fusion conjugate (or fusionmolecule). Fc fusion conjugates have been shown to increase the systemichalf-life of biopharmaceuticals, and thus the biopharmaceutical productmay require less frequent administration.

Fc binds to the neonatal Fc receptor (FcRn) in endothelial cells thatline the blood vessels, and, upon binding, the Fc fusion molecule isprotected from degradation and re-released into the circulation, keepingthe molecule in circulation longer. This Fc binding is believed to bethe mechanism by which endogenous IgG retains its long plasma half-life.More recent Fc-fusion technology links a single copy of abiopharmaceutical to the Fc region of an antibody to optimize thepharmacokinetic and pharmacodynamic properties of the biopharmaceuticalas compared to traditional Fc-fusion conjugates.

Other Modifications:

The present disclosure contemplates the use of other modifications,currently known or developed in the future, of the Polypeptides toimprove one or more properties. One such method for prolonging thecirculation half-life, increasing the stability, reducing the clearance,or altering the immunogenicity or allergenicity of a polypeptide of thepresent disclosure involves modification of the polypeptide sequences byhesylation, which utilizes hydroxyethyl starch derivatives linked toother molecules in order to modify the molecule's characteristics.Various aspects of hesylation are described in, for example, U.S. PatentAppln. Nos. 2007/0134197 and 2006/0258607.

Any of the foregoing components and molecules used to modify thepolypeptide sequences of the present disclosure may optionally beconjugated via a linker. Suitable linkers include “flexible linkers”which are generally of sufficient length to permit some movement betweenthe modified polypeptide sequences and the linked components andmolecules. The linker molecules are generally about 6-50 atoms long. Thelinker molecules may also be, for example, aryl acetylene, ethyleneglycol oligomers containing 2-10 monomer units, diamines, diacids, aminoacids, or combinations thereof. Suitable linkers can be readily selectedand can be of any suitable length, such as 1 (e.g., Gly), 2, 3, 4, 5, 6,7, 8, 9, 10, 10-20, 20-30, 30-50 amino acids (e.g., Gly).

Exemplary flexible linkers include glycine polymers (G)_(n),glycine-serine polymers (for example, (GS)_(n), GSGGS_(n) (SEQ ID NO:40)and GGGS_(n) (SEQ ID NO:41), where n is an integer of at least one),glycine-alanine polymers, alanine-serine polymers, and other flexiblelinkers. Glycine and glycine-serine polymers are relativelyunstructured, and therefore may serve as a neutral tether betweencomponents. Exemplary flexible linkers include, but are not limited toGGSG (SEQ ID NO:42), GGSGG (SEQ ID NO:43), GSGSG (SEQ ID NO:44), GSGGG(SEQ ID NO:45), GGGSG (SEQ ID NO:46), and GSSSG (SEQ ID NO:47).

Methods of Production of Polypeptides

A polypeptide of the present disclosure can be produced by any suitablemethod, including recombinant and non-recombinant methods (e.g.,chemical synthesis).

A. Chemical Synthesis

Where a polypeptide is chemically synthesized, the synthesis may proceedvia liquid-phase or solid-phase. Solid-phase peptide synthesis (SPPS)allows the incorporation of unnatural amino acids and/or peptide/proteinbackbone modification. Various forms of SPPS, such as Fmoc and Boc, areavailable for synthesizing polypeptides of the present disclosure.Details of the chemical synthesis are known in the art (e.g., Ganesan A.2006 Mini Rev. Med. Chem. 6:3-10; and Camarero J. A. et al., 2005Protein Pept Lett. 12:723-8).

Solid phase peptide synthesis may be performed as described hereafter.The α functions (Nα) and any reactive side chains are protected withacid-labile or base-labile groups. The protective groups are stableunder the conditions for linking amide bonds but can be readily cleavedwithout impairing the peptide chain that has formed. Suitable protectivegroups for the α-amino function include, but are not limited to, thefollowing: t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Z),o-chlorbenzyloxycarbonyl, bi-phenylisopropyloxycarbonyl,tert-amyloxycarbonyl (Amoc),α,α-dimethyl-3,5-dimethoxy-benzyloxycarbonyl, o-nitrosulfenyl,2-cyano-t-butoxy-carbonyl, 9-fluorenylmethoxycarbonyl (Fmoc),1-(4,4-dimethyl-2,6-dioxocylohex-1-ylidene)ethyl (Dde) and the like.

Suitable side chain protective groups include, but are not limited to:acetyl, allyl (All), allyloxycarbonyl (Alloc), benzyl (Bzl),benzyloxycarbonyl (Z), t-butyloxycarbonyl (Boc), benzyloxymethyl (Bom),o-bromobenzyloxycarbonyl, t-butyl (tBu), t-butyldimethylsilyl,2-chlorobenzyl, 2-chlorobenzyloxycarbonyl (2-CIZ), 2,6-dichlorobenzyl,cyclohexyl, cyclopentyl,1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde), isopropyl,4-methoxy-2,3-6-trimethylbenzylsulfonyl (Mtr),2,3,5,7,8-pentamethylchroman-6-sulfonyl (Pmc), pivalyl,tetrahydropyran-2-yl, tosyl (Tos), 2,4,6-trimethoxybenzyl,trimethylsilyl and trityl (Trt).

In the solid phase synthesis, the C-terminal amino acid is coupled to asuitable support material. Suitable support materials are those whichare inert towards the reagents and reaction conditions for the step-wisecondensation and cleavage reactions of the synthesis process and whichdo not dissolve in the reaction media being used. Examples ofcommercially-available support materials include styrene/divinylbenzenecopolymers which have been modified with reactive groups and/orpolyethylene glycol; chloromethylated styrene/divinylbenzene copolymers;hydroxymethylated or aminomethylated styrene/divinylbenzene copolymersand the like. Polystyrene (1%)-divinylbenzene or TentaGel® derivatizedwith 4-benzyloxybenzyl-alcohol (Wang-anchor) or 2-chlorotrityl chloridecan be used if it is intended to prepare the peptidic acid. In the caseof the peptide amide, polystyrene (1%) divinylbenzene or TentaGel®derivatized with 5-(4′-aminomethyl)-3′,5′-dimethoxyphenoxy)valeric acid(PAL-anchor) or p-(2,4-dimethoxyphenyl-amino methyl)-phenoxy group (Rinkamide anchor) can be used.

The linkage to the polymeric support can be achieved by reacting theC-terminal Fmoc-protected amino acid with the support material with theaddition of an activation reagent in ethanol, acetonitrile,N,N-dimethylformamide (DMF), dichloromethane, tetrahydrofuran,N-methylpyrrolidone or similar solvents at room temperature or elevatedtemperatures (e.g., between 40° C. and 60° C.) and with reaction timesof, e.g., 2 to 72 hours.

The coupling of the Nα-protected amino acid (e.g., the Fmoc amino acid)to the PAL, Wang or Rink anchor can, for example, be carried out withthe aid of coupling reagents such as N,N′-dicyclohexylcarbodiimide(DCC), N,N′-diisopropylcarbodiimide (DIC) or other carbodiimides,2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU) or other uronium salts, o-acyl-ureas,benzotriazol-1-yl-tris-pyrrolidino-phosphonium hexafluorophosphate(PyBOP) or other phosphonium salts, N-hydroxysuccinimides, otherN-hydroxyimides or oximes in the presence or also in the absence of1-hydroxybenzotriazole or 1-hydroxy-7-azabenzotriazole, e.g., with theaid of TBTU with addition of HOBt, with or without the addition of abase such as, for example, diisopropylethylamine (DIEA), triethylamineor N-methylmorpholine, e.g., diisopropylethylamine with reaction timesof 2 to 72 hours (e.g., 3 hours in a 1.5 to 3-fold excess of the aminoacid and the coupling reagents, e.g., in a 2-fold excess and attemperatures between about 10° C. and 50° C., e.g., 25° C. in a solventsuch as dimethylformamide, N-methylpyrrolidone or dichloromethane, e.g.,dimethylformamide).

Instead of the coupling reagents, it is also possible to use the activeesters (e.g., pentafluorophenyl, p-nitrophenyl or the like), thesymmetric anhydride of the Nα-Fmoc-amino acid, its acid chloride or acidfluoride under the conditions described above.

The Nα-protected amino acid (e.g., the Fmoc amino acid) can be coupledto the 2-chlorotrityl resin in dichloromethane with the addition of DIEAwith reaction times of 10 to 120 minutes, e.g., 20 minutes, but is notlimited to the use of this solvent and this base.

The successive coupling of the protected amino acids can be carried outaccording to conventional methods in peptide synthesis, typically in anautomated peptide synthesizer. After cleavage of the Nα-Fmoc protectivegroup of the coupled amino acid on the solid phase by treatment with,e.g., piperidine (10% to 50%) in dimethylformamide for 5 to 20 minutes,e.g., 2×2 minutes with 50% piperidine in DMF and 1×15 minutes with 20%piperidine in DMF, the next protected amino acid in a 3 to 10-foldexcess, e.g., in a 10-fold excess, is coupled to the previous amino acidin an inert, non-aqueous, polar solvent such as dichloromethane, DMF ormixtures of the two and at temperatures between about 10° C. and 50° C.,e.g., at 25° C. The previously mentioned reagents for coupling the firstNα-Fmoc amino acid to the PAL, Wang or Rink anchor are suitable ascoupling reagents. Active esters of the protected amino acid, orchlorides or fluorides or symmetric anhydrides thereof can also be usedas an alternative.

At the end of the solid phase synthesis, the peptide is cleaved from thesupport material while simultaneously cleaving the side chain protectinggroups. Cleavage can be carried out with trifluoroacetic acid or otherstrongly acidic media with addition of 5%-20% V/V of scavengers such asdimethylsulfide, ethylmethylsulfide, thioanisole, thiocresol, m-cresol,anisole ethanedithiol, phenol or water, e.g., 15% v/vdimethylsulfide/ethanedithiol/m-cresol 1:1:1, within 0.5 to 3 hours,e.g., 2 hours. Peptides with fully protected side chains are obtained bycleaving the 2-chlorotrityl anchor with glacial aceticacid/trifluoroethanol/dichloromethane 2:2:6. The protected peptide canbe purified by chromatography on silica gel. If the peptide is linked tothe solid phase via the Wang anchor and if it is intended to obtain apeptide with a C-terminal alkylamidation, the cleavage can be carriedout by aminolysis with an alkylamine or fluoroalkylamine. The aminolysisis carried out at temperatures between about −10° C. and 50° C. (e.g.,about 25° C.), and reaction times between about 12 and 24 hours (e.g.,about 18 hours). In addition the peptide can be cleaved from the supportby re-esterification, e.g., with methanol.

The acidic solution that is obtained may be admixed with a 3 to 20-foldamount of cold ether or n-hexane, e.g., a 10-fold excess of diethylether, in order to precipitate the peptide and hence to separate thescavengers and cleaved protective groups that remain in the ether. Afurther purification can be carried out by re-precipitating the peptideseveral times from glacial acetic acid. The precipitate that is obtainedcan be taken up in water or tert-butanol or mixtures of the twosolvents, e.g., a 1:1 mixture of tert-butanol/water, and freeze-dried.

The peptide obtained can be purified by various chromatographic methods,including ion exchange over a weakly basic resin in the acetate form;hydrophobic adsorption chromatography on non-derivatizedpolystyrene/divinylbenzene copolymers (e.g., Amberlite® XAD); adsorptionchromatography on silica gel; ion exchange chromatography, e.g., oncarboxymethyl cellulose; distribution chromatography, e.g., on Sephadex®G-25; countercurrent distribution chromatography; or high pressureliquid chromatography (HPLC) e.g., reversed-phase HPLC on octyl oroctadecylsilylsilica (ODS) phases.

B. Recombinant Production

Where a polypeptide is produced using recombinant techniques, thepolypeptide may be produced as an intracellular protein or as a secretedprotein, using any suitable construct and any suitable host cell, whichcan be a prokaryotic or eukaryotic cell, such as a bacterial (e.g., E.coli) or a yeast host cell, respectively. Other examples of eukaryoticcells that may be used as host cells include insect cells, mammaliancells, and/or plant cells. Where mammalian host cells are used, they mayinclude human cells (e.g., HeLa, 293, H9 and Jurkat cells); mouse cells(e.g., NIH3T3, L cells, and C127 cells); primate cells (e.g., Cos 1, Cos7 and CV1) and hamster cells (e.g., Chinese hamster ovary (CHO) cells).

A variety of host-vector systems suitable for the expression of apolypeptide may be employed according to standard procedures known inthe art. See, e.g., Sambrook et al., 1989 Current Protocols in MolecularBiology Cold Spring Harbor Press, New York; and Ausubel et al. 1995Current Protocols in Molecular Biology, Eds. Wiley and Sons. Methods forintroduction of genetic material into host cells include, for example,transformation, electroporation, conjugation, calcium phosphate methodsand the like. The method for transfer can be selected so as to providefor stable expression of the introduced polypeptide-encoding nucleicacid. The polypeptide-encoding nucleic acid can be provided as aninheritable episomal element (e.g., a plasmid) or can be genomicallyintegrated. A variety of appropriate vectors for use in production of apolypeptide of interest are commercially available.

Vectors can provide for extrachromosomal maintenance in a host cell orcan provide for integration into the host cell genome. The expressionvector provides transcriptional and translational regulatory sequences,and may provide for inducible or constitutive expression where thecoding region is operably-linked under the transcriptional control ofthe transcriptional initiation region, and a transcriptional andtranslational termination region. In general, the transcriptional andtranslational regulatory sequences may include, but are not limited to,promoter sequences, ribosomal binding sites, transcriptional start andstop sequences, translational start and stop sequences, and enhancer oractivator sequences. Promoters can be either constitutive or inducible,and can be a strong constitutive promoter (e.g., T7).

Expression constructs generally have convenient restriction siteslocated near the promoter sequence to provide for the insertion ofnucleic acid sequences encoding proteins of interest. A selectablemarker operative in the expression host may be present to facilitateselection of cells containing the vector. Moreover, the expressionconstruct may include additional elements. For example, the expressionvector may have one or two replication systems, thus allowing it to bemaintained in organisms, for example, in mammalian or insect cells forexpression and in a prokaryotic host for cloning and amplification. Inaddition, the expression construct may contain a selectable marker geneto allow the selection of transformed host cells. Selectable genes arewell known in the art and will vary with the host cell used.

Isolation and purification of a protein can be accomplished according tomethods known in the art. For example, a protein can be isolated from alysate of cells genetically modified to express the proteinconstitutively and/or upon induction, or from a synthetic reactionmixture by immunoaffinity purification, which generally involvescontacting the sample with an anti-protein antibody, washing to removenon-specifically bound material, and eluting the specifically boundprotein. The isolated protein can be further purified by dialysis andother methods normally employed in protein purification methods. In oneembodiment, the protein may be isolated using metal chelatechromatography methods. Proteins may contain modifications to facilitateisolation.

The polypeptides may be prepared in substantially pure or isolated form(e.g., free from other polypeptides). The polypeptides can be present ina composition that is enriched for the polypeptide relative to othercomponents that may be present (e.g., other polypeptides or other hostcell components). For example, purified polypeptide may be provided suchthat the polypeptide is present in a composition that is substantiallyfree of other expressed proteins, e.g., less than 90%, less than 60%,less than 50%, less than 40%, less than 30%, less than 20%, less than10%, less than 5%, or less than 1%, of the composition is made up ofother expressed proteins.

Antibodies

The present disclosure provides antibodies, including isolatedantibodies, that specifically bind a GDF15 polypeptide, e.g., a GDF15mutein of the present disclosure. The term “antibody” encompasses intactmonoclonal antibodies, polyclonal antibodies, multispecific antibodies(e.g., bispecific antibodies) formed from at least two intactantibodies, and antibody binding fragments including Fab and F(ab)′₂,provided that they exhibit the desired biological activity. The basicwhole antibody structural unit comprises a tetramer, and each tetrameris composed of two identical pairs of polypeptide chains, each pairhaving one “light” chain (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. In contrast, the carboxy-terminal portion of eachchain defines a constant region primarily responsible for effectorfunction. Human light chains are classified as kappa and lambda, whereashuman heavy chains are classified as mu, delta, gamma, alpha, orepsilon, and define the antibody's isotype as IgM, IgD, IgA, and IgE,respectively. Binding fragments are produced by recombinant DNAtechniques, or by enzymatic or chemical cleavage of intact antibodies.Binding fragments include Fab, Fab′, F(ab′)₂, Fv, and single-chainantibodies.

Each heavy chain has at one end a variable domain (VH) followed by anumber of constant domains. Each light chain has a variable domain atone end (VL) and a constant domain at its other end; the constant domainof the light chain is aligned with the first constant domain of theheavy chain, and the light chain variable domain is aligned with thevariable domain of the heavy chain. Within light and heavy chains, thevariable and constant regions are joined by a “J” region of about 12 ormore amino acids, with the heavy chain also including a “D” region ofabout 10 more amino acids. The antibody chains all exhibit the samegeneral structure of relatively conserved framework regions (FR) joinedby three hyper-variable regions, also called“complementarity-determining regions” or “CDRs”. The CDRs from the twochains of each pair are aligned by the framework regions, enablingbinding to a specific epitope. From N-terminal to C-terminal, both lightand heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3and FR4.

An intact antibody has two binding sites and, except in bifunctional orbispecific antibodies, the two binding sites are the same. A bispecificor bifunctional antibody is an artificial hybrid antibody having twodifferent heavy/light chain pairs and two different binding sites.Bispecific antibodies can be produced by a variety of methods includingfusion of hybridomas or linking of Fab′ fragments.

As set forth above, binding fragments may be produced by enzymatic orchemical cleavage of intact antibodies. Digestion of antibodies with theenzyme papain results in two identical antigen-binding fragments, alsoknown as “Fab” fragments, and an “Fc” fragment which has noantigen-binding activity. Digestion of antibodies with the enzyme pepsinresults in a F(ab′)₂ fragment in which the two arms of the antibodymolecule remain linked and comprise two-antigen binding sites. TheF(ab′)₂ fragment has the ability to crosslink antigen.

As used herein, the term “Fab” refers to a fragment of an antibody thatcomprises the constant domain of the light chain and the CH1 domain ofthe heavy chain.

When used herein, the term “Fv” refers to the minimum fragment of anantibody that retains both antigen-recognition and antigen-bindingsites. In a two-chain Fv species, this region includes a dimer of oneheavy-chain and one light-chain variable domain in non-covalentassociation. In a single-chain Fv species, one heavy-chain and onelight-chain variable domain can be covalently linked by a flexiblepeptide linker such that the light and heavy chains can associate in a“dimeric” structure analogous to that in a two-chain Fv species. It isin this configuration that the three CDRs of each variable domaininteract to define an antigen-binding site on the surface of the VH-VLdimer. While the six CDRs, collectively, confer antigen-bindingspecificity to the antibody, even a single variable domain (or half ofan Fv comprising only three CDRs specific for an antigen) has theability to recognize and bind antigen.

When used herein, the term “complementarity determining regions” or“CDRs” refers to parts of immunological receptors that make contact witha specific ligand and determine its specificity.

The term “hypervariable region” refers to the amino acid residues of anantibody which are responsible for antigen-binding. The hypervariableregion generally comprises amino acid residues from a CDR and/or thoseresidues from a “hypervariable loop”.

As used herein, the term “epitope” refers to binding sites forantibodies on protein antigens. Epitopic determinants usually comprisechemically active surface groupings of molecules such as amino acids orsugar side chains, as well as specific three-dimensional structural andcharge characteristics. An antibody is said to bind an antigen when thedissociation constant is ≦1 μM, ≦100 nM, or ≦10 nM. An increasedequilibrium constant (“K_(D)”) means that there is less affinity betweenthe epitope and the antibody, whereas a decreased equilibrium constantmeans that there is more affinity between the epitope and the antibody.An antibody with a K_(D) of “no more than” a certain amount means thatthe antibody will bind to the epitope with the given K_(D) or morestrongly. Whereas K_(D) describes the binding characteristics of anepitope and an antibody, “potency” describes the effectiveness of theantibody itself for a function of the antibody. There is not necessarilya correlation between an equilibrium constant and potency; thus, forexample, a relatively low K_(D) does not automatically mean a highpotency.

The term “selectively binds” in reference to an antibody does not meanthat the antibody only binds to a single substance, but rather that theK_(D) of the antibody to a first substance is less than the K_(D) of theantibody to a second substance. An antibody that exclusively binds to anepitope only binds to that single epitope.

When administered to humans, antibodies that contain rodent (i.e.,murine or rat) variable and/or constant regions are sometimes associatedwith, for example, rapid clearance from the body or the generation of animmune response by the body against the antibody. In order to avoid theutilization of rodent-derived antibodies, fully human antibodies can begenerated through the introduction of human antibody function into arodent so that the rodent produces fully human antibodies. Unlessspecifically identified herein, “human” and “fully human” antibodies canbe used interchangeably. The term “fully human” can be useful whendistinguishing antibodies that are only partially human from those thatare completely, or fully, human. The skilled artisan is aware of variousmethods of generating fully human antibodies.

In order to address possible human anti-mouse antibody responses,chimeric or otherwise humanized antibodies can be utilized. Chimericantibodies have a human constant region and a murine variable region,and, as such, human anti-chimeric antibody responses may be observed insome patients. Therefore, it is advantageous to provide fully humanantibodies against multimeric enzymes in order to avoid possible humananti-mouse antibody or human anti-chimeric antibody responses.

Fully human monoclonal antibodies can be prepared, for example, by thegeneration of hybridoma cell lines by techniques known to the skilledartisan. Other preparation methods involve the use of sequences encodingparticular antibodies for transformation of a suitable mammalian hostcell, such as a CHO cell. Transformation can be by any known method forintroducing polynucleotides into a host cell, including, for example,packaging the polynucleotide in a virus (or into a viral vector) andtransducing a host cell with the virus (or vector) or by transfectionprocedures known in the art. Methods for introducing heterologouspolynucleotides into mammalian cells are well known in the art andinclude dextran-mediated transfection, calcium phosphate precipitation,polybrene-mediated transfection, protoplast fusion, electroporation,encapsulation of the polynucleotide(s) in liposomes, and directmicroinjection of the DNA into nuclei. Mammalian cell lines available ashosts for expression are well known in the art and include, but are notlimited to, CHO cells, HeLa cells, and human hepatocellular carcinomacells.

The antibodies can be used to detect a Polypeptide of the presentdisclosure. For example, the antibodies can be used as a diagnostic bydetecting the level of one or more Polypeptides of the presentdisclosure in a subject, and either comparing the detected level to astandard control level or to a baseline level in a subject determinedpreviously (e.g., prior to any illness).

Another embodiment of the present disclosure entails the use of one ormore human domain antibodies (dAb). dAbs are the smallest functionalbinding units of human antibodies (IgGs) and have favorable stabilityand solubility characteristics. The technology entails a dAb(s)conjugated to HSA (thereby forming a “AlbudAb”; see, e.g., EP1517921B,WO2005/118642 and WO2006/051288) and a molecule of interest (e.g., apolypeptide sequence of the present disclosure). AlbudAbs are oftensmaller and easier to manufacture in microbial expression systems, suchas bacteria or yeast, than current technologies used for extending theserum half-life of polypeptides. As HSA has a half-life of about threeweeks, the resulting conjugated molecule improves the half-life of themolecule of interest. Use of the dAb technology may also enhance theefficacy of the molecule of interest.

Therapeutic and Prophylactic Uses

The present disclosure provides methods for treating or preventinghyperglycemia, hyperinsulinemia, glucose intolerance, glucose metabolismdisorders, obesity and other body weight disorders, as well as othermetabolic and metabolic-associated diseases, disorders and conditions bythe administration of the Polypeptides, or compositions thereof, asdescribed herein. Such methods may also have an advantageous effect onone or more symptoms associated with a disease, disorder or conditionby, for example, decreasing the severity or the frequency of a symptom.

In order to determine whether a subject may be a candidate for thetreatment or prevention of hyperglycemia, hyperinsulinemia, glucoseintolerance, and/or glucose disorders by the methods provided herein,various diagnostic methods known in the art may be utilized. Suchmethods include those described elsewhere herein (e.g., fasting plasmaglucose (FPG) evaluation and the oral glucose tolerance test (oGTT)).

In order to determine whether a subject may be a candidate for thetreatment or prevention of a body weight disorder (e.g., obesity) by themethods provided herein, parameters such as, but not limited to, theetiology and the extent of the subject's condition (e.g., how overweightthe subject is compared to reference healthy individual) should beevaluated. For example, an adult having a BMI between ˜25 and ˜29.9kg/m² may be considered overweight (pre-obese), while an adult having aBMI of ˜30 kg/m² or higher may be considered obese. For subjects who areoverweight and/or who have poor diets (e.g., diets high in fat andcalories), it is common to initially implement and assess the effect ofmodified dietary habits and/or exercise regimens before initiating acourse of therapy comprising one or more of the Polypeptides of thepresent disclosure. As discussed herein, the Polypeptides can effectappetite suppression.

Pharmaceutical Compositions

The Modulators (e.g., Polypeptides) of the present disclosure may be inthe form of compositions suitable for administration to a subject. Ingeneral, such compositions are “pharmaceutical compositions” comprisingone or more Modulators and one or more pharmaceutically acceptable orphysiologically acceptable diluents, carriers or excipients. In certainembodiments, the Modulators are present in a therapeutically acceptableamount. The pharmaceutical compositions may be used in the methods ofthe present disclosure; thus, for example, the pharmaceuticalcompositions can be administered ex vivo or in vivo to a subject inorder to practice the therapeutic and prophylactic methods and usesdescribed herein.

The pharmaceutical compositions of the present disclosure can beformulated to be compatible with the intended method or route ofadministration; exemplary routes of administration are set forth herein.Furthermore, the pharmaceutical compositions may be used in combinationwith other therapeutically active agents or compounds (e.g., glucoselowering agents) as described herein in order to treat or prevent thediseases, disorders and conditions as contemplated by the presentdisclosure.

The pharmaceutical compositions typically comprise a therapeuticallyeffective amount of at least one of the Modulators (e.g., Polypeptides)contemplated by the present disclosure and one or more pharmaceuticallyand physiologically acceptable formulation agents. Suitablepharmaceutically acceptable or physiologically acceptable diluents,carriers or excipients include, but are not limited to, antioxidants(e.g., ascorbic acid and sodium bisulfate), preservatives (e.g., benzylalcohol, methyl parabens, ethyl or n-propyl, p-hydroxybenzoate),emulsifying agents, suspending agents, dispersing agents, solvents,fillers, bulking agents, detergents, buffers, vehicles, diluents, and/oradjuvants. For example, a suitable vehicle may be physiological salinesolution or citrate buffered saline, possibly supplemented with othermaterials common in pharmaceutical compositions for parenteraladministration. Neutral buffered saline or saline mixed with serumalbumin are further exemplary vehicles. Those skilled in the art willreadily recognize a variety of buffers that could be used in thepharmaceutical compositions and dosage forms. Typical buffers include,but are not limited to, pharmaceutically acceptable weak acids, weakbases, or mixtures thereof. As an example, the buffer components can bewater soluble materials such as phosphoric acid, tartaric acids, lacticacid, succinic acid, citric acid, acetic acid, ascorbic acid, asparticacid, glutamic acid, and salts thereof. Acceptable buffering agentsinclude, for example, a Tris buffer,N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),2-(N-Morpholino)ethanesulfonic acid (MES),2-(N-Morpholino)ethanesulfonic acid sodium salt (MES),3-(N-Morpholino)propanesulfonic acid (MOPS), andN-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS).

After a pharmaceutical composition has been formulated, it may be storedin sterile vials as a solution, suspension, gel, emulsion, solid, ordehydrated or lyophilized powder. Such formulations may be stored eitherin a ready-to-use form, a lyophilized form requiring reconstitutionprior to use, a liquid form requiring dilution prior to use, or otheracceptable form. In some embodiments, the pharmaceutical composition isprovided in a single-use container (e.g., a single-use vial, ampoule,syringe, or autoinjector (similar to, e.g., an EpiPen®)), whereas amulti-use container (e.g., a multi-use vial) is provided in otherembodiments. Any drug delivery apparatus may be used to deliver thePolypeptides, including implants (e.g., implantable pumps) and cathetersystems, both of which are well known to the skilled artisan. Depotinjections, which are generally administered subcutaneously orintramuscularly, may also be utilized to release the polypeptidesdisclosed herein over a defined period of time. Depot injections areusually either solid- or oil-based and generally comprise at least oneof the formulation components set forth herein. One of ordinary skill inthe art is familiar with possible formulations and uses of depotinjections.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension. This suspension may beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents mentioned herein. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butane diol. Acceptable diluents,solvents and dispersion media that may be employed include water,Ringer's solution, isotonic sodium chloride solution, Cremophor EL™(BASF, Parsippany, N.J.) or phosphate buffered saline (PBS), ethanol,polyol (e.g., glycerol, propylene glycol, and liquid polyethyleneglycol), and suitable mixtures thereof. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordiglycerides. Moreover, fatty acids such as oleic acid find use in thepreparation of injectables. Prolonged absorption of particularinjectable formulations can be achieved by including an agent thatdelays absorption (e.g., aluminum monostearate or gelatin).

The pharmaceutical compositions containing the active ingredient may bein a form suitable for oral use, for example, as tablets, capsules,troches, lozenges, aqueous or oily suspensions, dispersible powders orgranules, emulsions, hard or soft capsules, or syrups, solutions,microbeads or elixirs. Pharmaceutical compositions intended for oral usemay be prepared according to any method known to the art for themanufacture of pharmaceutical compositions, and such compositions maycontain one or more agents such as, for example, sweetening agents,flavoring agents, coloring agents and preserving agents in order toprovide pharmaceutically elegant and palatable preparations. Tablets,capsules and the like contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients which are suitable forthe manufacture of tablets. These excipients may be, for example,diluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia, and lubricating agents, for example magnesiumstearate, stearic acid or talc.

The tablets, capsules and the like suitable for oral administration maybe uncoated or coated by known techniques to delay disintegration andabsorption in the gastrointestinal tract and thereby provide a sustainedaction. For example, a time-delay material such as glyceryl monostearateor glyceryl distearate may be employed. They may also be coated bytechniques known in the art to form osmotic therapeutic tablets forcontrolled release. Additional agents include biodegradable orbiocompatible particles or a polymeric substance such as polyesters,polyamine acids, hydrogel, polyvinyl pyrrolidone, polyanhydrides,polyglycolic acid, ethylene-vinylacetate, methylcellulose,carboxymethylcellulose, protamine sulfate, or lactide/glycolidecopolymers, polylactide/glycolide copolymers, or ethylenevinylacetatecopolymers in order to control delivery of an administered composition.For example, the oral agent can be entrapped in microcapsules preparedby coacervation techniques or by interfacial polymerization, by the useof hydroxymethylcellulose or gelatin-microcapsules orpoly(methylmethacrolate) microcapsules, respectively, or in a colloiddrug delivery system. Colloidal dispersion systems include macromoleculecomplexes, nano-capsules, microspheres, microbeads, and lipid-basedsystems, including oil-in-water emulsions, micelles, mixed micelles, andliposomes. Methods of preparing liposomes are described in, for example,U.S. Pat. Nos. 4,235,871, 4,501,728, and 4,837,028. Methods for thepreparation of the above-mentioned formulations will be apparent tothose skilled in the art.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate, kaolin ormicrocrystalline cellulose, or as soft gelatin capsules wherein theactive ingredient is mixed with water or an oil medium, for examplepeanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture thereof. Such excipients can besuspending agents, for example sodium carboxymethylcellulose,methylcellulose, hydroxy-propylmethylcellulose, sodium alginate,polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing orwetting agents, for example a naturally-occurring phosphatide (e.g.,lecithin), or condensation products of an alkylene oxide with fattyacids (e.g., polyoxy-ethylene stearate), or condensation products ofethylene oxide with long chain aliphatic alcohols (e.g., forheptadecaethyleneoxycetanol), or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol (e.g.,polyoxyethylene sorbitol monooleate), or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides (e.g., polyethylene sorbitan monooleate). The aqueoussuspensions may also contain one or more preservatives.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified herein.

The pharmaceutical compositions of the present disclosure may also be inthe form of oil-in-water emulsions. The oily phase may be a vegetableoil, for example olive oil or arachis oil, or a mineral oil, forexample, liquid paraffin, or mixtures of these. Suitable emulsifyingagents may be naturally-occurring gums, for example, gum acacia or gumtragacanth; naturally-occurring phosphatides, for example, soy bean,lecithin, and esters or partial esters derived from fatty acids; hexitolanhydrides, for example, sorbitan monooleate; and condensation productsof partial esters with ethylene oxide, for example, polyoxyethylenesorbitan monooleate.

Formulations can also include carriers to protect the compositionagainst rapid degradation or elimination from the body, such as acontrolled release formulation, including implants, liposomes,hydrogels, prodrugs and microencapsulated delivery systems. For example,a time delay material such as glyceryl monostearate or glyceryl stearatealone, or in combination with a wax, may be employed.

The present disclosure contemplates the administration of the Modulatorsin the form of suppositories for rectal administration of the drug. Thesuppositories can be prepared by mixing the drug with a suitablenon-irritating excipient which is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials include, but are not limited to,cocoa butter and polyethylene glycols.

The Modulators contemplated by the present disclosure may be in the formof any other suitable pharmaceutical composition (e.g., sprays for nasalor inhalation use) currently known or developed in the future.

The concentration of a polypeptide or fragment thereof in a formulationcan vary widely (e.g., from less than about 0.1%, usually at or at leastabout 2% to as much as 20% to 50% or more by weight) and will usually beselected primarily based on fluid volumes, viscosities, andsubject-based factors in accordance with, for example, the particularmode of administration selected.

Routes of Administration

The present disclosure contemplates the administration of the disclosedModulators (e.g., Polypeptides), and compositions thereof, in anyappropriate manner. Suitable routes of administration include parenteral(e.g., intramuscular, intravenous, subcutaneous (e.g., injection orimplant), intraperitoneal, intracisternal, intraarticular,intraperitoneal, intracerebral (intraparenchymal) andintracerebroventricular), oral, nasal, vaginal, sublingual, intraocular,rectal, topical (e.g., transdermal), sublingual and inhalation.

Depot injections, which are generally administered subcutaneously orintramuscularly, may also be utilized to release the Modulatorsdisclosed herein over a defined period of time. Depot injections areusually either solid- or oil-based and generally comprise at least oneof the formulation components set forth herein. One of ordinary skill inthe art is familiar with possible formulations and uses of depotinjections.

Regarding antibodies, in an exemplary embodiment an antibody or antibodyfragment of the present disclosure is stored at 10 mg/ml in sterileisotonic aqueous saline solution for injection at 4° C. and is dilutedin either 100 ml or 200 ml 0.9% sodium chloride for injection prior toadministration to the subject. The antibody is administered byintravenous infusion over the course of 1 hour at a dose of between 0.2and 10 mg/kg. In other embodiments, the antibody is administered byintravenous infusion over a period of between 15 minutes and 2 hours. Instill other embodiments, the administration procedure is viasubcutaneous bolus injection.

Combination Therapy

The present disclosure contemplates the use of the Modulators (e.g.,Polypeptides) in combination with one or more active therapeutic agentsor other prophylactic or therapeutic modalities. In such combinationtherapy, the various active agents frequently have different mechanismsof action. Such combination therapy may be especially advantageous byallowing a dose reduction of one or more of the agents, thereby reducingor eliminating the adverse effects associated with one or more of theagents; furthermore, such combination therapy may have a synergistictherapeutic or prophylactic effect on the underlying disease, disorder,or condition.

As used herein, “combination” is meant to include therapies that can beadministered separately, for example, formulated separately for separateadministration (e.g., as may be provided in a kit), and therapies thatcan be administered together in a single formulation (i.e., a“co-formulation”).

In certain embodiments, the Modulators are administered or appliedsequentially, e.g., where one agent is administered prior to one or moreother agents. In other embodiments, the Modulators are administeredsimultaneously, e.g., where two or more agents are administered at orabout the same time; the two or more agents may be present in two ormore separate formulations or combined into a single formulation (i.e.,a co-formulation). Regardless of whether the two or more agents areadministered sequentially or simultaneously, they are considered to beadministered in combination for purposes of the present disclosure.

The Modulators of the present disclosure can be used in combination withother agents useful in the treatment, prevention, suppression oramelioration of the diseases, disorders or conditions set forth herein,including those that are normally administered to subjects sufferingfrom hyperglycemia, hyperinsulinemia, glucose intolerance, and otherglucose metabolism disorders.

The present disclosure contemplates combination therapy with numerousagents (and classes thereof), including 1) insulin, insulin mimetics andagents that entail stimulation of insulin secretion, includingsulfonylureas (e.g., chlorpropamide, tolazamide, acetohexamide,tolbutamide, glyburide, glimepiride, glipizide) and meglitinides (e.g.,repaglinide (PRANDIN) and nateglinide (STARLIX)); 2) biguanides (e.g.,metformin (GLUCOPHAGE)) and other agents that act by promoting glucoseutilization, reducing hepatic glucose production and/or diminishingintestinal glucose output; 3) alpha-glucosidase inhibitors (e.g.,acarbose and miglitol) and other agents that slow down carbohydratedigestion and consequently absorption from the gut and reducepostprandial hyperglycemia; 4) thiazolidinediones (e.g., rosiglitazone(AVANDIA), troglitazone (REZULIN), pioglitazone (ACTOS), glipizide,balaglitazone, rivoglitazone, netoglitazone, troglitazone, englitazone,ciglitazone, adaglitazone, darglitazone that enhance insulin action(e.g., by insulin sensitization), thus promoting glucose utilization inperipheral tissues; 5) glucagon-like-peptides including DPP-IVinhibitors (e.g., vildagliptin (GALVUS) and sitagliptin (JANUVIA)) andGlucagon-Like Peptide-1 (GLP-1) and GLP-1 agonists and analogs (e.g.,exenatide (BYETTA)); 6) and DPP-IV-resistant analogues (incretinmimetics), PPAR gamma agonists, dual-acting PPAR agonists, pan-actingPPAR agonists, PTP1B inhibitors, SGLT inhibitors, insulin secretagogues,RXR agonists, glycogen synthase kinase-3 inhibitors, immune modulators,beta-3 adrenergic receptor agonists, 11beta-HSD1 inhibitors, and amylinanalogues.

Furthermore, the present disclosure contemplates combination therapywith agents and methods for promoting weight loss, such as agents thatstimulate metabolism or decrease appetite, and modified diets and/orexercise regimens to promote weight loss.

The Modulators of the present disclosure may be used in combination withone or more other agent in any manner appropriate under thecircumstances. In one embodiment, treatment with the at least one activeagent and at least one Modulator of the present disclosure is maintainedover a period of time. In another embodiment, treatment with the atleast one active agent is reduced or discontinued (e.g., when thesubject is stable), while treatment with the Modulator of the presentdisclosure is maintained at a constant dosing regimen. In a furtherembodiment, treatment with the at least one active agent is reduced ordiscontinued (e.g., when the subject is stable), while treatment withthe Modulator of the present disclosure is reduced (e.g., lower dose,less frequent dosing or shorter treatment regimen). In yet anotherembodiment, treatment with the at least one active agent is reduced ordiscontinued (e.g., when the subject is stable), and treatment with theModulator of the present disclosure is increased (e.g., higher dose,more frequent dosing or longer treatment regimen). In yet anotherembodiment, treatment with the at least one active agent is maintainedand treatment with the Modulator of the present disclosure is reduced ordiscontinued (e.g., lower dose, less frequent dosing or shortertreatment regimen). In yet another embodiment, treatment with the atleast one active agent and treatment with the Modulator of the presentdisclosure are reduced or discontinued (e.g., lower dose, less frequentdosing or shorter treatment regimen).

Dosing

The Modulators (e.g., Polypeptides) of the present disclosure may beadministered to a subject in an amount that is dependent upon, forexample, the goal of the administration (e.g., the degree of resolutiondesired); the age, weight, sex, and health and physical condition of thesubject to be treated; the nature of the Modulator, and/or formulationbeing administered; the route of administration; and the nature of thedisease, disorder, condition or symptom thereof (e.g., the severity ofthe dysregulation of glucose/insulin and the stage of the disorder). Thedosing regimen may also take into consideration the existence, nature,and extent of any adverse effects associated with the agent(s) beingadministered. Effective dosage amounts and dosage regimens can readilybe determined from, for example, safety and dose-escalation trials, invivo studies (e.g., animal models), and other methods known to theskilled artisan.

In general, dosing parameters dictate that the dosage amount be lessthan an amount that could be irreversibly toxic to the subject (i.e.,the maximum tolerated dose, “MTD”) and not less than an amount requiredto produce a measurable effect on the subject. Such amounts aredetermined by, for example, the pharmacokinetic and pharmacodynamicparameters associated with absorption, distribution, metabolism, andexcretion (“ADME”), taking into consideration the route ofadministration and other factors.

An effective dose (ED) is the dose or amount of an agent that produces atherapeutic response or desired effect in some fraction of the subjectstaking it. The “median effective dose” or ED50 of an agent is the doseor amount of an agent that produces a therapeutic response or desiredeffect in 50% of the population to which it is administered. Althoughthe ED50 is commonly used as a measure of reasonable expectance of anagent's effect, it is not necessarily the dose that a clinician mightdeem appropriate taking into consideration all relevant factors. Thus,in some situations the effective amount is more than the calculatedED50, in other situations the effective amount is less than thecalculated ED50, and in still other situations the effective amount isthe same as the calculated ED50.

In addition, an effective dose of the Modulators of the presentdisclosure may be an amount that, when administered in one or more dosesto a subject, produces a desired result relative to a healthy subject.For example, an effective dose may be one that, when administered to asubject having elevated plasma glucose and/or plasma insulin, achieves adesired reduction relative to that of a healthy subject by at leastabout 10%, at least about 20%, at least about 25%, at least about 30%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, or more than 80%.

An appropriate dosage level will generally be about 0.001 to 100 mg/kgof patient body weight per day, which can be administered in single ormultiple doses. In some embodiments, the dosage level will be about 0.01to about 25 mg/kg per day, and in other embodiments about 0.05 to about10 mg/kg per day. A suitable dosage level may be about 0.01 to 25 mg/kgper day, about 0.05 to 10 mg/kg per day, or about 0.1 to 5 mg/kg perday. Within this range, the dosage may be 0.005 to 0.05, 0.05 to 0.5 or0.5 to 5.0 mg/kg per day.

For administration of an oral agent, the compositions can be provided inthe form of tablets, capsules and the like containing from 1.0 to 1000milligrams of the active ingredient, particularly 1.0, 3.0, 5.0, 10.0,15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0,500.0, 600.0, 750.0, 800.0, 900.0, and 1000.0 milligrams of the activeingredient. The Modulators may be administered on a regimen of, forexample, 1 to 4 times per day, and often once or twice per day.

The dosage of the Modulators of the present disclosure may be repeatedat an appropriate frequency, which may be in the range of once per dayto once every three months, depending on the pharmacokinetics of theModulators (e.g. half-life) and the pharmacodynamic response (e.g. theduration of the therapeutic effect of the Modulator). In someembodiments where the Modulator is an antibody or a fragment thereof, ora polypeptide or variants thereof, dosing is frequently repeated betweenonce per week and once every 3 months. In other embodiments, suchModulators are administered approximately once per month.

In certain embodiments, the dosage of the disclosed Modulators iscontained in a “unit dosage form”. The phrase “unit dosage form” refersto physically discrete units, each unit containing a predeterminedamount of a Modulator of the present disclosure, either alone or incombination with one or more additional agents, sufficient to producethe desired effect. It will be appreciated that the parameters of a unitdosage form will depend on the particular agent and the effect to beachieved.

Kits

The present disclosure also contemplates kits comprising the disclosedModulators (e.g., Polypeptides), and pharmaceutical compositionsthereof. The kits are generally in the form of a physical structurehousing various components, as described below, and may be utilized, forexample, in practicing the methods described above (e.g., administrationof a Modulator to a subject in need of restoring glucose homeostasis).

A kit can include one or more of the Modulators disclosed herein(provided in, e.g., a sterile container), which may be in the form of apharmaceutical composition suitable for administration to a subject. TheModulators can be provided in a form that is ready for use or in a formrequiring, for example, reconstitution or dilution prior toadministration. When the Modulators are in a form that needs to bereconstituted by a user, the kit may also include buffers,pharmaceutically acceptable excipients, and the like, packaged with orseparately from the Modulators. When combination therapy iscontemplated, the kit may contain the several agents separately or theymay already be combined in the kit. Each component of the kit can beenclosed within an individual container and all of the variouscontainers can be within a single package. A kit of the presentdisclosure can be designed for conditions necessary to properly maintainthe components housed therein (e.g., refrigeration or freezing).

A kit may contain a label or packaging insert including identifyinginformation for the components therein and instructions for their use(e.g., dosing parameters, clinical pharmacology of the activeingredient(s), including mechanism of action, pharmacokinetics andpharmacodynamics, adverse effects, contraindications, etc.). Labels orinserts can include manufacturer information such as lot numbers andexpiration dates. The label or packaging insert may be, e.g., integratedinto the physical structure housing the components, contained separatelywithin the physical structure, or affixed to a component of the kit(e.g., an ampoule, tube or vial). Exemplary instructions include thosefor reducing or lowering blood glucose, treatment of hyperglycemia,treatment of diabetes, etc. with the disclosed Modulators, andpharmaceutical compositions thereof

Labels or inserts can additionally include, or be incorporated into, acomputer readable medium, such as a disk (e.g., hard disk, card, memorydisk), optical disk such as CD- or DVD-ROM/RAM, DVD, MP3, magnetic tape,or an electrical storage media such as RAM and ROM or hybrids of thesesuch as magnetic/optical storage media, FLASH media or memory-typecards. In some embodiments, the actual instructions are not present inthe kit, but means for obtaining the instructions from a remote source,e.g., via the internet, are provided.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g., amounts, temperature, etc.), but someexperimental errors and deviations should be accounted for.

Unless indicated otherwise, parts are parts by weight, molecular weightis weight average molecular weight, temperature is in degrees Celsius (°C.), and pressure is at or near atmospheric. Standard abbreviations areused, including the following: bp=base pair(s); kb=kilobase(s);pl=picoliter(s); s or sec=second(s); min=minute(s); h or hr=hour(s);aa=amino acid(s); kb=kilobase(s); nt=nucleotide(s); ng=nanogram;μg=microgram; mg=milligram; g=gram; kg=kilogram; dl or dL=deciliter; μlor μL=microliter; ml or mL=milliliter; l or L=liter; μM=micromolar;mM=millimolar; M=molar; kDa=kilodalton; i.m.=intramuscular(ly);i.p.=intraperitoneal(ly); s.c.=subcutaneous(ly); bid=twice daily;HPLC=high performance liquid chromatography; BW=body weight; U=unit;ns=not statistically significant; PG=fasting plasma glucose; FPI=fastingplasma insulin; ITT=insulin tolerance test; PTT=pyruvate tolerance test;oGTT=oral glucose tolerance test; GSIS=glucose-stimulated insulinsecretion; PBS=phosphate-buffered saline; PCR=polymerase chain reaction;NHS=N-Hydroxysuccinimide; DMEM=Dulbeco's Modification of Eagle's Medium;GC=genome copy; EDTA=ethylenediaminetetraacetic acid.

Materials and Methods

The following methods and materials were used in the Examples below:

Animals.

Diet-induced obese (DIO) male C57BL/6J mice (The Jackson Laboratory, BarHarbor, Me.) were maintained on a high-fat diet (D12492, Research Diets,Inc, New Brunswick, N.J.) containing 60 kcal % fat, 20 kcal % proteinand 20 kcal % carbohydrate for 12-20 weeks. All animal studies wereapproved by the NGM Institutional Animal Care and Use Committee.

Nine-week old male B6.V-LEP^(ob)/J (leptin-deficient (ob/ob)) mice (TheJackson Laboratory, Bar Harbor, Me.) were used in the recombinant doingstudies. Mice had free access to autoclaved distilled water and were fedad libitum a commercial mouse chow (Irradiated 2018 Teklad Global 18%protein Rodent Diet, Harlan Laboratories, Dublin, Va.). All animalstudies were approved by the NGM Institutional Animal Care and UseCommittee.

Nucleic Acid and Amino Acid Sequences.

GenBank Accession No. BC000529.2 sets forth the cDNA of ORF encodinghuman GDF15, and GenBank Accession No. NP_(—)004855.2 sets forth theamino acid sequence encoded by the cDNA. The 112-amino acid GDF15 (FIG.1B), or a GDF15 mutein, was used.

GDF15 ORF was amplified with polymerase chain reaction (PCR) using arecombinant DNA (cDNA) clone purchased from Open Biosystems (Cat. #MHS1011-58735). PCR reagents kits with Phusion high-fidelity DNApolymerase were purchased from New England BioLabs (F-530L, Ipswich,Mass.). The following primers were used: forward PCR primer: 5′TGCTCTAGAATGCCCGGGCAAG (SEQ ID NO:38) and reverse PCR primer: 5′CCATCGATCTATCATATGCAGTGGCA (SEQ ID NO:39).

Amplified DNA fragment was digested with restriction enzymes Xba I andClaI (the restriction sites were included in the 5′ (Xba I) and 3′ (ClaI) PCR primers, respectively) and was then ligated with AAV transgenevectors that had been digested with the same restriction enzymes. Thevector used for expression contained a selectable marker and anexpression cassette composed of a strong eukaryotic promoter 5′ of asite for insertion of the cloned coding sequence, followed by a 3′untranslated region and bovine growth hormone polyadenylation tail. Theexpression construct is also flanked by internal terminal repeats at the5′ and 3′ ends.

Production and Purification of AAV.

293 cells (Agilent Technologies, Santa Clara, Calif.) were cultured inDMEM (Mediatech, Inc., Manassas, Va.) supplemented with 10% fetal bovineserum and 1× antibiotic-antimycotic solution (Mediatech, Inc., Manassas,Va.). On day 1, the cells were plated at 50% density in 150 mm cellculture plates. On day 2, the cells were transfected using calciumphosphate precipitation method with the following 3 plasmids (20μg/plate of each): AAV transgene plasmid, pHelper plasmids (AgilentTechnologies, Santa Clara, Calif.) and AAV2/9 plasmid (Gao et al., J.Virol. 78:6381 (2004)).

Forty-eight hours after transfection, the cells were scraped off theplates, pelleted by centrifugation at 3000×g and re-suspended in buffercontaining 20 mM Tris pH 8.5, 100 mM NaCl and 1 mM MgCl₂. The suspensionwas frozen in an alcohol dry ice bath and was then thawed in a 37° C.water bath; the freeze and thaw cycles were repeated three times.Benzenase (Sigma-aldrich, St. Louis, Mo.) was added to a finalconcentration of 50 units/ml, and deoxycholate was added to a finalconcentration of 0.25%. After incubation at 37° C. for 30 min, celldebris was pelleted by centrifugation at 5000×g for 20 min. Viralparticles in the supernatant were purified using a iodixanal(Sigma-aldrich, St. Louis, Mo. (discontinued)) gradient as previouslydescribed (Zolotukhin et al., Gene Ther. 6:973 (1999)). The viral stockwas concentrated using Vivaspin 20 (MW cutoff 100,000 Dalton, SartoriusStedim Biotech, Aubagne, France) and re-suspended in PBS with 10%glycerol and stored at −80° C. To determine the viral genome copynumber, 2 μl of viral stock were incubated in 6 μl of solutioncontaining 50 units/ml Benzonase, 50 mM Tris-HCl pH 7.5, 10 mM MgCl₂ and10 mM CaCl₂ at 37° C. for 30 minutes.

Thereafter, 15 μl of a solution containing 2 mg/ml of Proteinase K, 0.5%SDS and 25 mM EDTA were added and the mixture was incubated for anadditional 20 min at 55° C. to release viral DNA. Viral DNA was cleanedwith mini DNeasy Kit (Qiagen, Valencia, Calif.) and eluted with 40 μl ofwater. Viral GC was determined using quantitative PCR. Viral stock wasdiluted with PBS to desirable GC/ml. Viral working solution (200 μl) wasdelivered into mice via tail vein injection.

Blood Glucose Assay.

Blood glucose from mouse tail snip was measured using ACCU-CHEK Activetest strips read by ACCU-CHEK Active meter (Roche Diagnostics,Indianapolis, Ind.) following manufacturer's instruction.

Serum GDF15 Variants Exposure Level Assay.

Whole blood (˜50 μl/mouse) from mouse tail snips was collected intoplain capillary tubes (BD Clay Adams SurePrep, Becton Dickenson, Sparks,Md.). Serum and blood cells were separated by spinning the tubes in anAutocrit Ultra 3 (Becton Dickinson, Sparks, Md.). GDF15 exposure levelsin serum were determined using Human GDF-15 Quantikine ELISA Kit (R&DSystems, Minneapolis, Minn.) by following the manufacturer'sinstructions.

Pegylation of GDF15 Muteins.

NHS-specific chemistries were used to modify recombinant GDF15 muteins,at the N-terminus and/or internally in a site-specific manner, with alinear 10 kDa PEG moiety (Nanocs Inc.; New York, N.Y.; cat. no.PG1-SC-10k). When desired, prevention of PEGylation at the N-terminalalanine was effected using sulfo-NHS-acetate (Thermo Fisher Scientific;Waltham, Mass.; cat. no. 26777). Labeling reactions were followed as permanufacturer's instructions, and excess label was quenched using a 1/100(v/v) addition of 1M Tris pH 8.0.

Example 1 Effects of GDF15 on Body Weight and Fasted Blood Glucose inHigh-Fat Fed Diet Induced Obesity (DIO) Mice

To evaluate the effect of continuous GDF15 exposure on body weight andfasted glucose serum levels, 19-week old high-fat fed, DIO mice (n=7)weighing approximately 45 g and having four-hour fasted glucose serumlevels of approximately 237 mg/dl, received a single bolus tail veininjection of AAV virus containing the above-described GDF15 gene insert.Serum systemic exposure was monitored by Human GDF-15 Quantikine ELISAKit (R&D Systems, Minneapolis, Minn.) following the manufacturer'sinstructions (FIG. 3). The data in FIG. 3 demonstrate systemic exposurelevels of GDF15 in mice from genetic methods in order to establish aclear relationship of observed phenotype compared to GDF15 serumconcentration (see FIGS. 4-7). Using these exposure levels as abenchmark, mice in Example 3 were administered recombinant GDF15 atdoses that attained approximately equivalent serum level concentrationsto those observed via genetic validation.

As depicted in FIG. 4, systemic exposure of GDF15 for 2 weekssignificantly decreased body weight at serum concentrations above 14.9ng/mL. Following 2 weeks of systemic exposure, body weight decreases of5.9 g comprising a 12.4% decrease (**, p<0.01) and 8.2 g comprising a17.3% decrease (***, p<0.001) were observed for medium (14.9 ng/mL) andhigh (65.6 ng/mL) exposures respectively, relative to PBS-injected DIOmice at week 2. In each group of mice, n=7 and p-values were determinedby 2-way ANOVA comparing body weight at week 2 with PBS vehicle control(average body weight=47.7 g).

As depicted in FIG. 5, systemic exposure of GDF15 for 2 weekssignificantly decreased fasted serum glucose levels at all systemicserum concentrations of GDF15. Following two weeks of systemic exposure,4-hour fasted blood glucose level decreases of 31.3 mg/dl comprising a13.5% decrease (**, p<0.01), 52.0 mg/dl comprising a 22.5% decrease(***, p<0.001), and 53.3 mg/dl comprising a 23.0% decrease (***,p<0.001) were observed for low (0.7 ng/mL), medium (14.9 ng/mL) and high(65.6 ng/mL) exposures, respectively, relative to PBS-injected DIO miceat week 2. In each group of mice, n=7 and p-values were determined by1-way ANOVA comparing body weight at week 2 compared to PBS vehiclecontrol (average serum glucose level=231.3 mg/dl).

Example 2 Systemic Exposure Effects of GDF15 Muteins on Body Weight andFasted Blood Glucose in High-Fat Fed Diet-Induced Obese Mice

To evaluate the effect of GDF15 muteins on body weight and fastedglucose serum levels, 19-week old high-fat fed, DIO mice (weighing ˜41 gand having 4-hour fasted glucose serum levels of ˜218 mg/dl) received asingle bolus tail vein injection of medium dose AAV (14.9 ng/mL)containing the GDF15 mutein gene insert described above. Systemic serumexposures at were approximated to be equivalent to those of GDF15 atmedium dose as set forth in FIG. 3.

As depicted in FIG. 6, systemic exposure of GDF15 muteins for 2 weeksresulted in decreased body weight trends at medium AAV dose. Following 2weeks of systemic exposure, body weight decreases, relative toPBS-injected DIO mice at week 2, were as follows: GDF15 decrease of 6.1g (ns), v1 decrease of 5.9 g (ns), v2 decrease of 3.0 g (ns), v3decrease of 1.1 g (ns), v4 decrease of 1.5 g (ns), v5 decrease of 5.2 g(ns), v6 decrease of 5.3 g (ns) and v7 decrease of 6.2 g (ns). In eachgroup of mice, n=5 and p-values were determined by 1-way ANOVA comparingbody weight at week 2 following GDF15 mutein dosing with PBSvehicle-control dosing (average body weight=41.4 g).

As depicted in FIG. 7, systemic exposure of GDF15 muteins for 2 weekssignificantly decreased fasted serum glucose levels at medium AAV dose.Following two weeks of systemic exposure, 4-hour fasted serum glucoselevels, relative to PBS-injected DIO mice at week 2, were as follows:GDF15 decrease of 37 mg/dl (**, p<0.01), v1 decrease of 36.6 mg/dl (**,p<0.01), v2 decrease of 11.8 mg/dl (ns), v3 decrease of 15.3 mg/dl (ns),v4 decrease of 9.2 mg/dl (ns), v5 decrease of 30.8 mg/dl (*, p<0.05), v6decrease of 31 mg/dl (*, p<0.05) and v7 decrease of 31.8 mg/dl (*,p<0.05). In each group of mice, n=5 and p-values were determined by1-way ANOVA comparing four-hour fasted serum blood glucose at week 2following GDF15 mutein dosing with PBS vehicle-control dosing (averageblood glucose=229.2 mg/dl).

Example 3 Effect of Recombinant GDF15 on Food Intake, Body Weight andNon-Fasted Blood Glucose in Leptin-deficient ob/ob Mice

To evaluate the effect of dose-dependent recombinant GDF15 exposure onfood intake, body weight and fasted glucose serum levels, 9-week oldob/ob mice (three dosing groups, n=7 per group) weighing approximately44.5 g (average of all dosing groups) and having non-fasted glucoseserum levels of approximately 393.6 mg/dl (average of all dosinggroups), received recombinant GDF15 via s.c. bid delivery. Serum GDF15exposure was monitored by Human GDF-15 Quantikine ELISA Kit (R&DSystems, Minneapolis, Minn.) following the manufacturer's instructions.

On day one, the first dosing group received (w/w equivalent) 0.02 mg/kg,the second dosing group received 0.2 mg/kg, and the third dosing groupreceived 2 mg/kg. Referring to FIG. 8, the serum exposure (measured fourhours after administration of the last (second) dose) is represented bythe unshaded bar (first dosing group), grey shaded bar (second dosinggroup), and black shaded bar (highest dosing group). From day 2 to day7, the first dosing group received (w/w equivalent) 0.01 mg/kg twicedaily, the second dosing group received 0.1 mg/kg twice daily, and thethird dosing group received 1 mg/kg twice daily; the serum exposure(measured four hours after administration of the last (second) dose onday 7) is represented by the unshaded bar (first dosing group), greyshaded bar (second dosing group), and black shaded bar (third dosinggroup).

As indicated in FIG. 8, exposure to recombinant GDF15 was within theequivalent range (ng/mL) of that established in Example 1 (see FIG. 3).These data establish a correlation of equivalency between serum GDF15levels and body weight and blood glucose phenotypes for both the geneticand the recombinant GDF15 modalities.

The dose-dependent effect of recombinant GDF15 on food intake(grams/day/animal) was then determined during the course of one week oftwice-daily s.c. dosing. On day one, singly housed mice were fasted inthe morning and the first dosing group received (w/w equivalent) 0.02mg/kg, the second dosing group received 0.2 mg/kg, and the third dosinggroup received 2 mg/kg. Eight hours later this dosing regimen wasrepeated. Thereafter, food was returned back to the animal cages. On themorning of day 2, the remaining food was weighed and each animal'sovernight (“O/N”) food intake was calculated. For day 2 to day 7, thefirst dosing group received (w/w equivalent) 0.01 mg/kg, the seconddosing group received 0.1 mg/kg and the third dosing group received 1mg/kg. Each animal's food intake from day 2 to day 4 and from day 4 today 7 was again measured. The food intake measurement from day 2 to day7 was made prior to each day's dose.

Referring to FIG. 9, food intake in vehicle-control animals isrepresented by the unshaded bars, while the effect on food intake on thefirst dosing group is represented by the light-grey shaded bars, on thesecond dosing group is represented by the medium-grey shaded bars, andon the third dosing group is represented by the black shaded bars. Asdepicted in FIG. 9, recombinant GDF15 significantly decreased foodintake relative to vehicle control-injected ob/ob mice at days 1, 4 and7. For day 1, food intake was as follows: vehicle control dosinggroup=5.3 g, first dosing group=4.9 g (ns), second dosing group=4.3 g(*, p<0.05) and third dosing group=3.7 g (***, p<0.001). For day 2 today 4, food intake was as follows: vehicle control dosing group=7.6 g;first dosing group=6.4 g (*, p<0.05); second dosing group=5.5 g (***,p<0.001); and third dosing group=3.7 g (***, p<0.001). For day 4 to day7, food intake was as follows: vehicle control dosing group=7.7 g; firstdosing group=6.7 g (ns); second dosing group=5.3 g (***, p<0.001); andthird dosing group=4.3 g (***, p<0.001). The average food intake(grams/day/animal) for the duration of the experiment for all timepoints (days 1-7) was as follows: vehicle control dosing group=7.2 g;first dosing group=6.3 g (ns); second dosing group=5.2 g (***, p<0.001);and third dosing group=4.5 g (***, p<0.001). In each group of mice, n=7and p-values were determined by 2-way ANOVA comparing food intake ateach time point compared to vehicle control.

As depicted in FIG. 10, mice injected with recombinant GDF15 bid s.c.demonstrated significantly decreased body weight compared to vehiclecontrol-injected ob/ob mice at days 1, 4 and 7. On day one, the firstdosing group received (w/w equivalent) 0.02 mg/kg, the second dosinggroup received 0.2 mg/kg, and the third dosing group received 2 mg/kg.For day 2 to day 7, the first dosing group received (w/w equivalent)0.01 mg/kg, the second dosing group received 0.1 mg/kg and the thirddosing group received 1 mg/kg. On each day, body weight was recordedprior to the morning dose.

For day 1, body weight was as follows: vehicle-control dosing group=44.8g; first dosing group=44.9 g (ns); second dosing group=44.2 g (ns); andthird dosing group=44.7 g (ns). For day 4, body weight was as follows:vehicle-control dosing group=47.1 g; first dosing group=46.4 g (ns);second dosing group=44.2 g (ns); and third dosing group=44.6 g (ns). Forday 7, body weight was as follows: vehicle-control dosing group=48.2 g;first dosing group=47.0 g (ns); second dosing group=44.2 g (**, p<0.01);and third dosing group=44.0 g (**, p<0.01). In each group of mice, n=7and p-values were determined by 2-way ANOVA comparing body weight ateach time point compared to vehicle control.

As depicted in FIG. 11, s.c. bid exposure of recombinant GDF15 resultedin significantly decreased non-fasted blood glucose serum levels 16hours post-dose relative to vehicle control-injected ob/ob mice at days4 and 7. Mice were injected with vehicle (shaded circles), 0.02 mg/kg(first dosing group; shaded squares), 0.2 mg/kg (second dosing group;shaded triangles) or 2 mg/kg (third dosing group; unshaded circles). Forday 2 to day 7, the first dosing group received (w/w equivalent) 0.01mg/kg, the second dosing group received 0.1 mg/kg and the third dosinggroup received 1 mg/kg. At days 4 and 7, the second and third dosinggroups exhibited significantly decreased non-fasted blood glucose serumlevels (mg/dl) relative to vehicle control-injected levels. For day 1,glucose levels were as follows: vehicle control=392.4 mg/dl; firstdosing group=395.0 mg/dl (ns); second dosing group=395.4 mg/dl (ns); andthird dosing group=391.7 mg/dl (ns). For day 4, glucose levels were asfollows: vehicle control=422.1 mg/dl; first dosing group=381.3 mg/dl(ns); second dosing group=301.9 mg/dl (**, p<0.01); and third dosinggroup=247.7 mg/dl (***, p<0.001). For day 7, glucose levels were asfollows: vehicle control=455.6 mg/dl; first dosing group=389.7 mg/dl(ns); second dosing group=304.1 mg/dl (***, p<0.001); and third dosinggroup=204.7 mg/dl (***, p<0.001). In each group of mice, n=7 andp-values were determined by 2-way ANOVA comparing non-fasted bloodglucose at each time point compared to vehicle control.

Example 4 Effect of PEGylated Recombinant GDF15 Muteins on Food Intakeand Body Weight in Leptin-Deficient ob/ob Mice

The effect of recombinant, site-specifically PEGylated GDF15 muteins onfood intake and body weight was compared to that of the correspondingnon-PEGylated GDF15 muteins. Using the methods described above, variant1 was PEGylated at the N-terminus (v1-PEG10) (SEQ ID NO:5); variant 5was PEGylated at lysine69 (v5-PEG10) (SEQ ID NO:9); and variant 6 wasPEGylated at lysine91 (v6-PEG10) (SEQ ID NO:10). Ten-week old ob/ob micereceived a single s.c. dose of vehicle; non-PEGylated (2 mg/kg) v1, v5or v6; or PEGylated (2 mg/kg) v1, v5 or v6 (v1-PEG10, v5-PEG10, andv6-PEG10, respectively).

As depicted in FIG. 12, administration of a single dose of recombinantGDF15 muteins resulted in significantly decreased food intake, relativeto vehicle control-injected ob/ob mice, at days 1, 2, 5-7 and 15. Forday 1, food intake measured 16 hours following the first dose wasfollows (grams/day/animal): vehicle control 5.1 g, v1 2.5 g (***,p<0.001), v5 4.3 g (ns), v6 3.4 g (***, p<0.001), v1-PEG10 3.2 g (***,p<0.001), v5-PEG10 3.5 g (***, p<0.001), and v6-PEG10 3.2 g (***,p<0.001). Measurements made during the day 2-day 15 time period weretaken at approximately the same time (9:30 a.m.) each day. For day 2,food intake was as follows (grams/day/animal): vehicle control 5.7 g, v15.1 g (ns), v5 5.6 g (ns), v6 5.7 g (ns), v1-PEG10 3.8 g (***, p<0.001),v5-PEG10 4.3 g (**, p<0.01), and v6-PEG10 4.7 g (ns). For days 5-7, theaverage daily food intake was as follows (grams/day/animal): vehiclecontrol 5.4 g, v1 5.5 g (ns), v5 6.0 g (ns), v6 6.0 g (ns), v1-PEG10 3.5g (***, p<0.001), v5-PEG10 4.0 g (***, p<0.001), and v6-PEG10 4.2 g (**,p<0.01). For day 15, food intake was as follows (grams/day/animal):vehicle control 5.3 g, v1 5.3 g (ns), v5 5.6 g (ns), v6 5.8 g (ns),v1-PEG10 5.2 g (ns), v5-PEG10 4.9 g (ns), and v6-PEG10 5.1 g (ns). Ineach group of mice, n=5, and p-values were determined by one-way ANOVAcomparing food intake at each time point with PBS vehicle control.

As depicted in FIG. 13, administration of a single dose of recombinantGDF15 muteins resulted in significantly decreased body weight, relativeto PBS vehicle control-injected ob/ob mice, at specific time points ondays 1, 2, 8 and 15 respectively. For day 1, body weight measured 16hours following the first dose was as follows: vehicle control 49.3 g,v1 48.7 g (ns), v5 47.5 g (ns), v6 49.9 g (ns), v1-PEG10 46.8 g (ns),v5-PEG10 46.9 g (ns) and v6-PEG10 48.6 g (ns). Measurements made duringthe day 2-day 15 time period were taken at approximately the same time(9:30 a.m.) each day. For day 2, body weight was as follows: vehiclecontrol 51.0 g, v1 49.1 g (ns), v5 48.5 g (ns), v6 50.7 g (ns), v1-PEG1047.3 g (*, p<0.05), v5-PEG10 47.7 g (ns), and v6-PEG10 49.4 g (ns). Forday 8, body weight was as follows: vehicle control 52.9 g, v1 51.7 g(ns), v5 50.2 g (ns), v6 53.4 g (ns), v1-PEG10 45.8 g (***, p<0.001),v5-PEG10 47.3 g (***, p<0.001), and v6-PEG10 48.7 g (**, p<0.01). Forday 15, body weight was as follows: vehicle control 54.8 g, v1 54.0 g(ns), v5 52.0 g (ns), v6 55.5 g (ns), v1-PEG10 48.8 g (***, p<0.001),v5-PEG10 50.6 g (**, p<0.01) and v6-PEG10 52.4 (ns). In each group ofmice, n=5, and p-values were determined by two-way ANOVA comparing bodyweight at each time point with PBS vehicle control.

Particular embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Upon reading the foregoing, description, variations of the disclosedembodiments may become apparent to individuals working in the art, andit is expected that those skilled artisans may employ such variations asappropriate. Accordingly, it is intended that the invention be practicedotherwise than as specifically described herein, and that the inventionincludes all modifications and equivalents of the subject matter recitedin the claims appended hereto as permitted by applicable law. Moreover,any combination of the above-described elements in all possiblevariations thereof is encompassed by the invention unless otherwiseindicated herein or otherwise clearly contradicted by context.

All publications, patent applications, accession numbers, and otherreferences cited in this specification are herein incorporated byreference as if each individual publication or patent application werespecifically and individually indicated to be incorporated by reference.

1. A peptide comprising any one of: a) a peptide comprising at least onemodification to the polypeptide depicted in FIG. 1B (SEQ ID NO:3),wherein the modification does not alter the amino acid sequence of thepolypeptide; b) a mutein peptide of the sequence depicted in FIG. 1B(SEQ ID NO:3); or c) a mutein peptide of the sequence depicted in FIG.1B (SEQ ID NO:3), wherein the mutein peptide comprises at least onemodification that does not alter the amino acid sequence of the muteinpeptide.
 2. The peptide of claim 1, wherein the peptide comprises amutein peptide of any one of v1-v23 as depicted in FIG.
 2. 3. Thepeptide of claim 1, wherein the at least one modification comprisespegylation, glycosylation, polysialylation, hesylation, albumin fusion,albumin binding through a conjugated fatty acid chain, Fc-fusion, orfusion with a PEG mimetic.
 4. The peptide of claim 3, wherein the atleast one modification comprises pegylation.
 5. The peptide of claim 4,wherein the pegylation increases solubility of the peptide.
 6. Thepeptide of claim 1, wherein the mutein peptide has at least 85% aminoacid identity to the sequence depicted in FIG. 1B (SEQ ID NO:3).
 7. Thepeptide of claim 1, wherein the mutein peptide has at least 90% aminoacid identity to the sequence depicted in FIG. 1B (SEQ ID NO:3).
 8. Thepeptide of claim 1, wherein the mutein peptide has at least 95% aminoacid identity to the sequence depicted in FIG. 1B (SEQ ID NO:3).
 9. Thepeptide of claim 1, wherein the peptide has a length of from about 10amino acids to about 113 amino acids.
 10. The peptide of claim 1,wherein the peptide has fewer than 75 amino acid residues.
 11. Thepeptide of claim 1, wherein the peptide has fewer than 50 amino acidresidues.
 12. The peptide of claim 1, wherein the peptide has fewer than25 amino acid residues.
 13. The peptide of claim 1, wherein the peptideis produced recombinantly.
 14. A nucleic acid molecule encoding thepeptide of claim
 1. 15. The nucleic acid molecule of claim 14, whereinthe nucleic acid molecule is operably linked to an expression controlelement that confers expression of the nucleic acid molecule encodingthe peptide in vitro, in a cell, or in vivo.
 16. A vector comprising thenucleic acid molecule of claim
 14. 17. The vector of claim 16, whereinthe vector comprises a viral vector.
 18. A transformed or host cell thatexpresses the peptide of claim
 1. 19. A pharmaceutical composition,comprising a peptide of claim 1, and a pharmaceutically acceptablediluent, carrier or excipient.
 20. The pharmaceutical composition ofclaim 19, further comprising at least one additional prophylactic ortherapeutic agent.
 21. An antibody that binds specifically to a muteinpeptide of claim
 1. 22. A sterile container comprising thepharmaceutical composition of claim
 19. 23. The sterile container ofclaim 22, wherein the sterile container is a syringe.
 24. A kitcomprising the sterile container of claim
 22. 25. The kit of claim 24,further comprising a second sterile container comprising at least oneadditional prophylactic or therapeutic agent.
 26. A method of treatingor preventing a glucose metabolism disorder or a body weight disorder ina subject, comprising administering to the subject a therapeuticallyeffective amount of a peptide of claim
 1. 27. The method of claim 26,wherein the peptide comprises a mutein peptide of any one of v1-v23 asdepicted in FIG.
 2. 28. The method of claim 26, wherein the at least onemodification to the peptide comprises pegylation, glycosylation,polysialylation, hesylation, albumin fusion, albumin binding through aconjugated fatty acid chain, Fc-fusion, or fusion with a PEG mimetic.29. The method of claim 28, wherein the at least one modificationcomprises pegylation.
 30. The method of claim 29, wherein the pegylationincreases solubility.
 31. The method of claim 26, wherein the treatingor preventing comprises a reduction in blood glucose in the subject. 32.The method of claim 26, wherein the treating or preventing comprises areduction in body weight in the subject.
 33. The method of claim 26,wherein the treating or preventing comprises a reduction in food intakein the subject.
 34. The method of claim 26, wherein the glucosemetabolism disorder is diabetes mellitus.
 35. The method of claim 26,wherein the subject is human.
 36. The method of claim 26, wherein thesubject is obese.
 37. The method of claim 26, wherein the administeringis by parenteral injection.
 38. The method of claim 26, wherein theparenteral injection is subcutaneous.